Theranostics 2012, 2(2) 156

Ivyspring

International Publisher Theranostics 2012; 2(2):156-178. doi: 10.7150/thno.4068 Review -Activated Drug Development Ki Young Choi1,3, Magdalena Swierczewska1,2,3, Seulki Lee1, Xiaoyuan Chen1

1. Laboratory of Molecular Imaging and Nanomedicine (LOMIN), National Institute of Biomedical Imaging and Bioengi- neering (NIBIB), National Institutes of Health (NIH), Bethesda, Maryland, 20892, USA 2. Department of Biomedical Engineering, Stony Brook University, Stony Brook, NY 11794, USA 3. These authors are contributed equally.

 Corresponding author: E-mail: [email protected] and [email protected]

© Ivyspring International Publisher. This is an open-access article distributed under the terms of the Creative Commons License (http://creativecommons.org/ licenses/by-nc-nd/3.0/). Reproduction is permitted for personal, noncommercial use, provided that the article is in whole, unmodified, and properly cited.

Received: 2012.01.10; Accepted: 2012.01.28; Published: 2012.02.08

Abstract In this extensive review, we elucidate the importance of and their role in drug development in various diseases with an emphasis on cancer. First, key proteases are in- troduced along with their function in disease progression. Next, we link these proteases as targets for the development of prodrugs and provide clinical examples of protease-activatable prodrugs. Finally, we provide significant design considerations needed for the development of the next generation protease-targeted and protease-activatable prodrugs.

Key words: Protease, activatable probe, Alzheimer’s disease, cancer, caspase, cathepsin, kallikrein, MMP, PSA, , aspartyl protease

1. Introduction Proteases play a fundamental and essential role influential roles in DNA replication and transcription, in many biological and pathological processes by the cell proliferation and differentiation, angiogenesis, regulatory mechanism, proteolysis. Proteolysis is an neurogenesis, ovulation, fertilization, wound repair, irreversible regulatory mechanism and now known to stem cell mobilization, hemostasis, blood coagulation, selectively cleave specific substrates. Additionally, inflammation, immunity, senescence, necrosis and multimeric and multicatalytic proteases exist to de- apoptosis [4]. Therefore, deregulated modifications in grade multiple intracellular proteins, called pro- proteolytic actions underlie many diseases like cancer teasomes, essential for biological processes [1]. The and neurodegenerative and cardiovascular disorders. human degradome, which makes up a complete list of Because of proteases’ ability to degrade extracellular proteases synthesized by human cells, is made up of matrices and proteins, they are strongly associated at least 569 proteases that are distributed into five with cancer progression, specifically invasion and broad classes (in order from greatest to least number): metastasis. Additionally, intracellular proteases, like , serine, cysteine, threonine, and lysosomal cysteine proteases, are involved in more aspartic proteases [2]. Serine, cysteine and threonine protective mechanisms, like degrading many endo- proteases are involved in covalent catalysis. The nu- cytosed proteins and foreign bodies. cleophile of the catalytic site is part of the specified With strong evidence of protease involvement in . Metalloproteinases and aspartic proteases diseases, proteases serve an important role in drug perform non-covalent catalysis and the nucleophile is development. Some therapies have been formulated an activated water molecule [3]. to target and inhibit proteases and proteasomes that By their highly controlled actions, proteases play are dysregulated, especially for tumor suppression.

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Proteasome inhibitors have shown success in treat- stability, permeability and distribution [12-13]. Since ment of haematological malignancies and have prodrugs can overcome major hurdles of drug for- therefore been tested as therapeutic agents in the mulations like poor solubility or instability, previ- clinic for over 10 years [5]. The first inhibitor, borte- ously unsuitable drugs for clinical use can now be zomib has been used as a treatment for relapsed mul- utilized. It has been estimated that about 5-7% of tiple myeloma and mantle cell lymphoma. However, drugs currently approved worldwide are classified as the use of such expansive protease inhibitors have prodrugs and that an even larger amount of prodrugs shown a lack of success overall. For a comprehensive are approved every year [14]. Prodrugs are made up review of clinical successes and failures of protease of the parent drug conjugated with a promoiety, like a inhibitors see reference [3] and of proteasome inhibi- polymer or substrate via a cleavable linkage tors see reference [5]. Therefore, more specific prote- and/or a targeting moiety for specific delivery like an ase-inspired therapies have been attempted. First, the antibody or aptamer. Common functional groups design of recombinant forms of proteases can replace used to modify prodrugs for superior properties, defective protease but are limited by the large doses called promoieties, are listed in ref. [12]. In this re- necessary to achieve this effect. Second, gene-therapy view, we will focus on cleavable prodrugs, approaches targeting protease genes can intrinsically specifically protease-cleavable. Using this strategy, improve proper protease activity. This approach has the prodrug only achieves its active form when the been show to work in lentiviral-mediated enzyme of interest, for which the promoiety is its gene transfer to block prostate cancer growth [6]. substrate, cleaves it. Therefore, the drug is released at Third, an indirect approach is to hamper protease a specific location where the enzyme is overexpressed. inhibitors to reduce protective (anti-tumor) proteases. This review will focus on the promising devel- In this way, proteases are activated that can sensitize opment of PAPs. We will introduce the key target cancer cells to drug treatments or induce apoptosis, as proteases and their involvement in specific diseases seen by Karikari, et al. for the inhibition of caspase and further discuss the development of preclinical inhibitors to induce apoptosis in pancreatic cancer [7]. and clinical prodrugs utilizing these proteases. Final- Fourth, proteases can serve as biomarkers for diagno- ly, we will give a perspective on design considerations sis or prognosis of tumors. The presence of protective to develop advanced, efficient PAPs. The review will proteases can predict good clinical prognosis but their give the reader a background on the important pro- absence can indicate the need for different treatments. teases involved in various diseases, the design and Dysregulated proteolytic activities can signify the outcomes of prodrugs used to target and treat these progression of disease. An effective detection tech- diseases and an outlook of how this field can advance nique is the use of protease activatable probes [8-11]. further. Activatable probes, or molecular beacons, can signal, typically by fluorescence, the detection of proteases 2. Target proteases after the protease degrades the linkage between the Aberrant protease signaling pathways lead to dye and a quencher. In other words, the probe in its cancer as well as neurodegenerative, cardiovascular natural form gives off no signal; but only once a spe- and pulmonary diseases. Specific substrates for up- cific protease is present and degrades its specific sub- regulated proteases can be used as a promoiety for strate, the probe is activated. Using a similar ap- prodrugs, where the substrate is catalyzed to activate proach, protease-activated prodrugs (PAPs) can be the prodrug into therapeutics. Currently, a large exploited to improve drug delivery to areas where number of proteases have been identified as bi- protease expression, like in malignant tissues, is omarkers for early diagnostic and prognostic markers, higher than in normal tissues. especially for cancer. Here we will discuss which Prodrugs are derivatives of drug molecules that proteases are particularly identified in pathological can undergo a transformation by an enzyme, chemical disorders and their substrates (Table 1 and 2). A pro- or environmental stimuli to release the active parent tease substrate contains a recognition sequence for the drug in vivo [12]. Just as activatable probes, prodrugs protease to cleave. But in order to be used in prodrug in their native state are inactive forms of the drug but design, the substrate must reach the same location as only after a stimuli release an active drug. In this way, the protease [3]. prodrugs are an extremely efficient approach to in- In terms of cancer, dysregulated intracellular crease selectivity and efficacy of chemotherapy, re- proteases can cause a loss of protective mechanisms ducing the toxic effects on healthy cells. By chemical and cause overgrowth and overexpression of extra- conjugation, prodrugs improve all pharmaceutical cellular proteases, which has been shown to result in properties of the parent drug, such as its solubility, tumor metastasis. Currently, it is believed that uro-

http://www.thno.org Theranostics 2012, 2(2) 158 kinase plasminogen activator (uPA), cathepsin B, and healthy cells [16]. Therefore the development of PAPs membrane-type matrix (MMP) can allows for specific, active drug delivery to cancer sites. initiate the activation of pro-MMPs. Then, extracellu- Proteases are produced not only by tumor cells but lar matrix (ECM) degrading activities begin by extra- also by multiple cell types recruited to the tumor site cellular serine proteases, like uPA, urokinase plas- [18]. Therefore, the protease substrate selected in the minogen activator receptor (uPAR), plasminogen, and prodrug can be catalyzed directly in the tumor mi- MMPs to initiate cellular motility, invasiveness and a croenvironment to reduce non-specific toxicity in further cascade of tumor growth factors [15-17]. Re- normal proliferating and healthy cells. Here we in- cently, work has shown that certain extracellular troduce key proteases linked in cancer progression – proteases have anti-tumor properties [2]. Yet, over- cathepsins, kallikreins, uPA, uPAR, caspase and ma- expressed proteases have been identified in cancerous trix metalloproteinases. Then we summarize their cells numerous concentration folds higher than in roles in other diseases.

Table 1. Target proteases and diseases associated with overexpressed proteases

Family Protease Location Cancer Ref. Other Diseases Ref. Intracellular, Table in General Most lysosomes [121] Extracellular, Artherosclerosis, Cathepsin K Breast [178] [179-182] bone osteoporosis Extracellular Breast, cervix, colon, Cysteine and colorectal, gastric, head and Cathepsins pericellular [31, 38, 81, Cathepsin B neck, liver, lung, melanoma, under 183-196] ovarian, pancreatic, prostate, pathological thyroid conditions Cathepsin L Breast, colorectal [28] AD [197] Endosomal Cervical, gastric, lung, Cathepsin E structures, ER, [51-55] Aspartic pancreas adenocarcinomas Golgi Cathepsins [47-49, 198- Cathepsin D Lysosome Breast, colorectal, ovarian Atherosclerosis [121] 200] Intracellular, Table in [15, General Most secreted 58] Hypertension, hK1 [24] inflammation Kallikreins (hK) PSA (hK 3) Prostate, ovarian [201-202] Colon, ovarian, pancreatic, hK10 [203-206] head and neck hK15 Ovarian, prostate [207-208] Serine Membrane, Cervical, colorectal, gastric, [86, 116, uPA, uPAR Proteases Pericellular prostate 209-210] Neurodegenerative Caspases Intracellular [82] disorders Table in General Extracellular Most [211] [85, 102-104, MMP-1, -8, -13 Breast Artherosclerosis, RA [213-214] 211-212] Bronchiectasis, chronic MMPs asthma, COPD, cystic Breast, colorectal, lung, [91-94] [95- [87, 113- MMP-2, -9 fibrosis, HIV malignant gliomas, ovarian 98] 117] associated dementia, hypertension, stroke MMP-14 Membrane Breast [212] [105, 107, ADAM Extracellular AD 112] *Abbreviations : AD: Alzheimer’s disease; ADAM: a disintegrin and metalloproteinase domain protease; COPD: chronic obstructive pulmonary disease; ER: endoplasmic reticulum; RA: rheumatoid arthritis

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Table 2. Protease-activatable prodrugs

Protease Construct Drug Disease Ref.

Peptide-based prodrug Asp-Glu-Val-Asp-Pro-PABC-X Caspase-3 DOX, PH-A Cancer [215-217] CAR-Lys-Gly-Ser-Gly-Asp-Val-Glu-Gly-X Cancer, Cathepsin B N-L-Leu-X DNR, DOX [124-130] RA CP Arg-X, Ala-X, Asp-X MTX Cancer [218]

FAP BHQ3-Lys-Gln-Glu-Gln-Asn-Pro-Gly-Ser-Thr-X PH-A Cancer [219]

Kallikrein 2 Gly-Lys-Ala-Phe-Arg-Arg-X TPG Cancer [220, 221]

MMP-2/-9/-14 Glu-Pro-Cit-Gly-Hof-Tyr-Leu-X DOX Cancer [157, 158] BHQ3-Lys-Arg-Ala-Leu-Gly-Leu-Pro-Gly-X MMP-7 PH-A Cancer [222, 223] BHQ3-(D-Glu)8-Arg- Pro-Leu-Ala-Leu-Trp-Arg-Ser-(D-Arg)8-Lys-X D-Ala-Phe-Lys-X ara-C, AT-125, Cancer, Plasmin D-Val-Leu-Lys-X [224-230] DOX, PM RA D-Ala-Phe-Lys-(PABC)n-X Mu-His-Ser-Ser-Lys-Leu-Gln-Leu-X Mu-His-Ser-Ser-Lys-Leu-Gln-EDA-X DOX, 5-FudR, [148-153, PSA 4-O-(Ac-Hyp-Ser-Ser-Chg-Gln-Ser-Ser-Pro)-X VNB, TPG, Cancer 231-234] HO2C(Ch2)3CO-Hyp-Ala-Ser-Chg-Gln-Ser-Leu-X L12ADT N-glutaryl-(4-hydroxyprolyl)-Ala-Ser-chGly-Gln-Ser-Leu-X TOP β-Ala-L-Leu-L-Ala-L-Leu-X DOX Cancer [235-239] uPA D-Ala-Phe-Lys-PABC-X DOX Cancer [224, 225] Macromolecular prodrug PEG-L-lys-X Ce6, DNR, poly-L-glutamic acid-X DOX, 5-FU, [131, 134- Cathepsin B HPMAcp-Gly-Phe-Leu-Gly-X, MTX, PtD, Cancer 146, 240- ALB-Lys-Lys-Phe-D-Ala-EMC-X PTX, SN-392, 242] ALB-EMC-D-Ala-Phe-Lys-Lys-X TNP-470 Bone Cathepsin K HPMAcp-Gly-Gly-Pro-Nle-4AB-X ALN, PGE1 [243-245] disease DEX-Gly-Ile-Leu-Gly-Val-Pro-X [159-161, MMP-2/-9 DOX, MTX Cancer ALB-Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln-X 246] Cancer, Plasmin ALB-EMC-D-Ala-Phe-Lys-Lys-X DOX [242, 247] RA ALB-EMC-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly-X PSA DOX, TPG Cancer [154-156] HPMAcp-morpholinocarbonyl-Ser-Ser-Lys-Tyr-Gln-Leu-X PEG-poly-L-lysine-X Cancer, Thrombin Ce6, PH-A [248-250] poly-L-lysin-Gly-D-Phe-Pip-Arg-Ser-Gly-Gly-Gly-Gly-Gly-X RA poly-L-lysine-X Trypsin PH-A Cancer [251, 252] poly-L-lysine-Gly-Ala-Ser-D-Arg-Phe-Thr-Gly-X uPA ALB-EMC-Gly-Gly-Gly-Arg-Arg-X DOX Cancer [253]

Targeted prodrug c1F6-Val-Cit-X cAC10-Val-Cit-X DOX, MMAE, [164, 175, Cathepsin B Cancer Pep42-Val-Cit-X PTX 254-257] GAL-HPMAcp-Gly-Phe-Leu-Gly-X Cancer, Plasmin RGD-4C-D-Ala-Phe-Lys-(PABC) -X MTX [172] n RA

* Abbreviations : 4-AB: 4-aminobenzyl alcohol, ALB: albumin, ALN: alendronate, ara-C: 1-8-D-arabinofuranosylcytosine, AT-125: α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid, BHQ3: black hole quencher 3, cAC10: chimeric anti-CD30 monoclonal antibody, CAR: carotenoid, Ce6: chlorine e6, c1F6: chimeric anti-CD70 monoclonal antibody, chGly: cyclohexaglycyl, Cit: citrulline, CPT: camptothecin, CP: , dAc: desacetyl, DEX: dextran, DNR: daunorubicin, DOX: doxorubicin, DTX: docetaxel, EMC: ε-maleimidocaproic acid, FAP: fibroblast activation protein, 5-FU: 5-fluorouracil, 5-FudR: 5-fluorodeoxyuridine, GAL: galactose, Hof: homophenylalanine, HPMAcp: N-(2-hydroxypropyl)methacrylamide copolymer, Hyp: trans-4-hydroxyproline, L12ADT: 8-O-(12[L-leucinoylamino]dodecanoyl)-8-O-debutanoylthapsigargin, MTX: metotrexate, MMAE: monomethyl auristatin E, MMP: , Mu: morpholinocarbonyl, Nle: norleucyl, PABC: para aminobenzyloxycarboxyl, PEG: polyethylene glycol, Pep42: a cyclic 13-mer oligopeptide, PH-A: pheophorabide a, Pip: piperidine, PM: N,N-bis(2-chloroethyl)-p-phenylenediamine (phenylenediamine mustard), PSA: pros- tate-specific antigen, PTX: paclitaxel, PtD: platinum-based drug, PGE1: prostaglandin E1, RA: rheumatoid arthritis, RGD-4C: bicyclic Cys-Asp-Cys-Arg-Gly-Asp-Cys-Phe-Cys, SN-392: 10-amino-7-hydroxy camptothecin, TNP-470: O-(chloracetyl-carbamoyl) fumagillol, TOP: thimet oligopepti- dase, TPG: thapsigargin, VNB: vinblastine, X: therapeutic agent or its analogue

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2.1. Cathepsins apoptosis [43-44]. Some cathepsins have exhibited protective roles [2], like cathepsin L [45]. Therefore, Cysteine cathepsins are primarily intracellular, prodrug activation by cathepsins may prove to be a lysosomal proteases that are responsible for protein good approach for anticancer drug delivery over turnover but have shown to be upregulated in cancer, therapies inhibiting proteases, which are relevant for often in early lesions [19]. Cathepsin-catalyzed sub- normal cell function. Overexpression of the cysteine strate cleavage usually involves nucleophilic cysteine cathepsin is well-documented in many aggressive , histidine, and an aspartate in the and forms of cancer [46], thereby giving drug-cathepsin cleavage is favored by acidic conditions [16]. Howev- substrate conjugates an opportunity to be activated in er, specific substrates of cysteine cathepsins involved cancerous environments and delivering chemothera- in pathological conditions need to be determined [19]. peutics directly to the site of need. Overexpressed cysteine cathepsins are implicated in Cathepsin E and lysosomal cathepsin D are ho- glioma [20-21], melanoma [22], cancer of the esopha- mologous aspartic proteases that play an equally im- gus[23], stomach [24], colon [25], prostate [26-27], portant role in protein turnover in the cell as cysteine breast [28] and lung [29] and have been used as cathepsins. Aspartic proteases are made up of 14 pathological biomarkers [30]. Active cysteine cathep- families but all usually exist in highly acidic condi- sins are most commonly found in the acidic condi- tions, like in lysosomes or in the digestive tract. Ca- tions of lysosomes with some exceptions. Cathepsin K thepsin D is found in the lysosome and is implicated is normally excreted to the extracellular space be- in prostate [47], breast [48] and colorectal cancer [49]. tween osteoclasts and bone for bone remodeling Cathepsin D expression and secretion parallel many [31-32]. Although intracellular proteases are less of the lysosomal cysteine proteases mentioned above. studied in their role in cancer compared to the more Cathepsin E, on the other hand, is not found in lyso- common ECM-degrading proteases like MMP and somes but in the intracellular space like endosomal uPA, deregulation in their locations can initiate fur- structures, endoplasmic reticulum and Golgi appa- ther extracellular proteolytic cascades [33-34]. For ratus, as well as in the plasma membrane and pre- example, secreted cathepsin B and L hydrolyze type dominantly associated with immune cells [50]. Ca- IV collagen, fibronectin [35], cell-adhesion proteins thepsin E overexpression has been associated in sev- [36] and laminin [35] to enable tumor cell proliferation eral forms of cancer like human gastric [51], cervical and instigate pro-MMPs. Cysteine cathepsins can also [52], pancreatic [53-54] adenocarcinomas and lung degrade collagen by intracellular proteolysis. Colla- carcinomas [55]. To date, most developed PAPs focus gen can be taken up by macrophage and tumor cells on cathepsin B because of their expression in the in- via uPA and uPAR and then degraded in the lyso- tracellular lysosomes. somes by cysteine cathepsins [19]. The most re- 2.2 Kallikrein and other serine proteases searched cathepsins, cathepsins B and L, are shown to be overexpressed via gene amplification, transcript Kallikreins are serine proteases that are tradi- variants expressed by tumor cells, transcription fac- tionally linked to poor clinical prognosis of human tors and post-transcriptional regulation [37]. Cathep- carcinoma. The most popular is prostate specific an- sin B has been identified in membrane caveolae, out- tigen (PSA), also known as human kallikrein 3 (hK3) side of the lysosome, of human colon cancer cells and that serves as a diagnostic biomarker for prostate hypothesized to mediate further cell-surface and ECM cancer. Serine proteases mediate substrate cleavage by degrading proteolytic events [38]. Recent work has histidine, serine and aspartic acid amino acids, which shown that the permeabilization of the lysosomal are usually close to one another and hKs additionally membrane is implicated in cancer, mostly due to the contain 10-12 cysteine residues. Tissue kallikrein gene discharge of many cysteine cathepsins from their in- (KLK) expression is highly regulated by sex-steroid nate location [39]. Once the cathepsins exit the cell and hormones [15], like androgen regulation of KLK2 and into the extracellular environment of tumors they can KLK3 [56], but there are also post-translational regu- be activated by the acidic conditions found in the lations to control the irreversible protease action. tumor microenvironment [40]. However, different Proteolytic activity of hKs can be inhibited by interac- mechanisms of activation are also possible. Cathepsin tions with plasma globulin, serpins, tissue inhibitors, K is shown to be activated by glycosaminoglycans ions [57] as well as by its own fragmentation [15]. while cathepsins B, L and S are secreted in active form The pro-enzyme can be activated by intracellular or [41]. Interestingly, cysteine cathepsins, like cathepsins extracellular hydrolysis or by other hKs [15]. Kal- B, H, L, S, and K [42], translocate to the intracellular likreins are found in sweat, milk, saliva, seminal without membrane association to initiate plasma and cerebrospinal fluid in humans because

http://www.thno.org Theranostics 2012, 2(2) 161 they are mainly secreted from epithelial cells in skin, cleavage of its extracellular segment and transmit breast, prostate, pancreas and brain. Under diseased intracellular signals to stimulate cancer cell growth conditions like cancer, hKs are dysregulated. In ovar- [76-78]. A comprehensive review on hKs and its role ian cancer, for example, twelve KLK genes are up- in cancer with tabulated clinical implications are regulated [15]. However, emerging data indicates that found in references [15] and [58], respectively. hKs can both promote and inhibit tumor progression 2.3 Caspases and is most likely dependent on hormone balances as well as the tissue type. This also makes determining Caspases are intracellular cysteine proteases that its substrate a challenge as it may be tissue specific are fundamental in – apopto- [58]. Otin et al. discusses the tumor suppressing roles sis [79]. They are one of the more specific and efficient of hK3, hK8, hK9, hK10, hK13, hK 14 [2]. Yet, it is the proteases [80]. Cancer cells can suppress caspase ex- overexpressed hKs in different cancers that can serve pression to circumvent apoptosis in order to prolifer- as viable targets for drug delivery. ate. Yet, some aggressive tumors undergo spontane- A variety of hKs have been implicated in cancer ous apoptosis [81]. Inhibitors of apoptosis proteins are progression like angiogenesis, invasion and metasta- regulators of the caspase cascade, specifically caspase sis, especially with its interaction with other serine 3 and 7, and have a large role in cancer [82]. Addi- proteases such as uPA and uPAR. But no hK has got- tionally, genetic factors such as deletion of caspase 8 ten as much attention as PSA (hK3) and hK2 in their and 10 genes and mutations in caspase 3, 5, 6 and 7 are role in prostate cancer. PSA can promote prostate associated with human tumors [2]. Caspases can serve tumor growth by numerous proposed pathways like as interesting proteases to study the mechanisms of the initiation of growth factors and proteolytic cas- cell death and a key hallmark of cancer, but its sup- cades to degrade the ECM. PSA and hK2 are identi- pression rather than overexpression is what governs fied as insulin-like growth factor (IGF) binding pro- cancer progression. tein proteases [59]. When they degrade the binding 2.4 Matrix metalloproteinases protein in the tumor microenvironment, they in turn The most established extracellular and pericel- increase the bioavailability of IGF. The growth factor lular proteases identified in cancer metastasis and can then easily stimulate the growth of prostate can- many other diseases are matrix metalloproteinases cer cells. Additionally, hK2 and hK4 can activate the (MMPs). MMPs make up a versatile family of prote- uPA proteolytic cascade by inactivating the plasmin- ases that control many physiological functions [83]. ogen activator inhibitor 1 [60-62]. Without the inhibi- But as seen in the previous sections, MMPs are acti- tor, uPA can bind to its receptor, uPAR, and convert vated by many other proteases and serve as the pro- plasminogen to plasmin, a serine protease. Plasmin teolytic endpoint for tumor progression. As their then leads to ECM degradation by activation of name suggest, MMPs modulate and regulate the pro-MMPs and release of growth factors, like endo- ECM. They are associated with different stages of thelial growth factor to promote angiogenesis [63]. cancer invasion and metastasis by releasing cancer Yet, the angiogenic properties of PSA are still debated; cells to spread and provide room for cancer cells to works show that PSA can activate tumor growth fac- invade. Additionally, ECM remodeling is involved in tor β (TFGβ) to promote angiogenesis [64] but also can releasing many important proteins such as cytokines, block fibroblast growth factor 2 and inhibit angio- growth factors and chemokines, and in turn MMPs genesis [65]. Furthermore, hKs can directly activate regulate many of these proteins. MMPs can be identi- ECM degrading MMPs, like type IV [66]. fied by the presence of Zn2+ ions at the catalytic center The uPA-uPAR pathway is a well studied mechanism that is coordinated by three histidine residues and a of cancer progression [67-69], and offers many op- serine residue. They are mainly excreted as an inactive portunities to target proteases like PSA, hK2, uPA, proenzyme and activated by a cysteine switch mech- plasmin and MMPs, for potential anti-cancer therapy. anism to expose the catalytic zinc pocket [84]. The Elevated levels of uPA and its related serine proteases main activation system is by the removal of the pro- have been observed in various cancers like colorectal domain of MMPs. As described above, proteases such [70], gastric [71], prostate [72], and cervical cancer as plasmin, serine proteases and other MMPs can [73]. Protease-activated receptor (PAR) signaling is proteolytically remove the prodomain of MMPs to another pathway that has been implicated in various activate them in the extracellular space [85]. Consist- cancers. PAR is a G-protein coupled receptor that is ing of at least 23 known human , the MMP expressed in many cancer cells and cells in the mi- family can be divided into five sub-groups: Colla- croenvironment of a tumor. It can be activated by genases: MMP-1, -8, -13; : MMP-2, -9; serine proteases like trypsin [74] and thrombin [75] by

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Stromelysins: MMP-3, -10, -11; Membrane-type MMPs required for normal cell function, their alterations can (MT-MMPs): MMP-15, -16, -17, -24, -25 and the re- also lead to disorders beyond cancer. Proteases have maining proteases make up a varied group. The fam- been implicated in diseases such as neurodegenera- ily is versatile and some MMPs have shown protective tive, pulmonary and cardiovascular. effects like MMP-8, -12,-26 and tumorigenic effects Proteases are getting increasing attention in their like MMP-9, -11 and more. role in Alzheimer’s disease (AD), a neurodegenerative Additionally MMPs have been implicated in disorder that affects approximately 2% of the popula- regulatory functions like survival, angiogenesis, in- tion in industrialized countries [105]. The pathological flammation and signaling as well as other pathologies phenotype of AD is characterized by amyloid plague like multiple sclerosis, stroke, Alzheimer’s disease, build up and neurofibrillary tangles in various areas lung emphysema, arthritis and infections of the cen- of the brain [106]. These plagues are believed to occur tral nervous system [86]. The versatility in MMPs due to elevated levels of neurotoxic amyloid beta comes from their ability to cleave multiple types of protein that can damage synapses and neuritis [105]. elements. For example the MMP-2 can The proteolytic processing of the amyloid beta protein cleave gelatin, type I, IV and I collagens, elastin, and from amyloid precursor protein (APP) is the pre- vitronectin [87]. MMP-9 can also degrade similar dominant abnormality in AD [105]. APP is a large substrates as MMP-2, except collagen I, and release glycoprotein that can be cleaved and activated to am- vascular endothelial growth factor to induce neovas- yloid beta protein by two aspartic proteases, cularization. Gelatinases, MMP-2 [88] and -9 [89], are β-secretase at the N-terminus and γ-secretase at the implicated in angiogenesis. Many papers have related C-terminus. Additionally, α-secretase, a disintegrin gelatinases to tumor metastasis and angiogenesis be- and metalloproteinase domain protease (ADAM), is cause of their ability to degrade the vascular basal known to cleave APP within the important amyloid membrane to induce growth of new blood vessels as beta protein sequence [105]. The ADAM family (made well as release growth factors responsible for tumor up of 21 known human ADAMs [107]) has adhesive growth [90]. Increased expression of gelatinase has and proteolytic properties, which can aid in mediat- been observed in many various cancers such as breast ing interaction with other molecules as well as sig- [91-93], ovarian [94], lung [95] and colorectal [96] as naling. This family is an emerging field and more re- well as malignant gliomas [97-98]. MMP-2 and -9 can search is needed to implicate these MMP-type prote- be activated by MMP-7, a protease that by itself is ases in various pathologies. An inhibitory and im- associated with cancer cell growth because of its abil- munization treatment has shown that a removal of ity to cleave insulin-like growth factor-binding pro- amyloid beta protein deposits can recover cognitive tein. In addition, MMP-7 is a special MMP because it defects associated with AD [108-109]. Therefore, pro- can be produced by cancerous cells [99], which in turn teases that are directly associated with amyloid further induces cancer invasiveness by activating plaque formation in AD can serve as promising ther- other metastasis associated proteases [100]. Although apeutic targets. Several proteases have shown to be MMP-14 is a membrane-type MMP, it is also associ- elevated in AD, but their use as diagnostic or prog- ated with similar substrates as gelatinases and release nostic biomarkers are still in preliminary stages of vascular endothelial growth factor. Furthermore, it [110-111]. Additionally, elevated levels of other MMP aids in decreasing cell adhesion by cleaving mem- types have been seen in AD [112] along with varying brane proteins like integrins, E-cadherin, and cell neurological conditions like stroke (MMP-9) [113-114] surface proteoglycans. MMPs make up a versatile and HIV associated dementia (MMP-2, -7, -9) [115]. family with broad substrate specificity that are Beyond MMPs, caspase overexpression has also been strongly associated with tumor progression. Although implicated in neurodegenerative disorders [82]. many treatments involving MMP inhibitors have Because of MMPs’ role in ECM remodeling and historically failed [101], increased MMP expression at angiogenesis, their dysregulation can lead to pulmo- tumor sites can play an important role in drug deliv- nary and cardiovascular disorders. The gelatinase ery. Excellent reviews on MMPs and their clinical family of MMPs (MMP-2, -9), are implicated in many implications are found in references [85, 102-104]. fibrous pulmonary diseases like chronic asthma [116], 2.5 Proteases in neurodegenerative, pulmonary cystic fibrosis, bronchiectasis, and chronic obstructive and cardiovascular diseases pulmonary disease (COPD) [87]. This may be due to dysregulation of ECM remodeling leading to a thick- As seen above, irregular protease signaling leads ening of the basement membrane. Protease signaling to dysregulation of homeostasis and can lead to can- pathway is involved in the blood coagulation cascade cer. Since proteases are regulatory enzymes and are and deregulation of proteases in the pathway can lead

http://www.thno.org Theranostics 2012, 2(2) 163 to cardiovascular disease. The specific roles that sorption, unwanted rapid metabolism or low selec- gelatinases play in angiogenesis has lead others to tivity; and also iii) reduce undesirable irritation, pain hypothesize their role in vasoconstrictive properties or non-specific toxicity to normal tissues [12-13]. and disorders such as hypertension [117]. Angioten- Prodrugs have minimal pharmacological effects sin-converting enzyme (ACE) is a metalloproteinase in its native state. However, pharmacological activity that is central to the blood coagulation cascade by is recovered when they are transformed into the ac- converting I to angiotensin II, required for tive parent drug or its derivative by disease-specific or angiotensin receptor activation and vessel constriction environmental stimuli. Among various targets of [118]. The use of ACE inhibitors have been used on prodrugs, proteases are considered an important tar- the market for over 20 years to treat hypertension, get because proteases are highly involved in diseases heart failure and heart attack [119]. Outside of the as described in Section 2 above. For PAPs to be effec- MMP family, hK1 is implicated in kinin production, tive, the linkage between the drug and promoiety which can mediate many biological functions like should remain stable in the bloodstream but degrade inflammation and hypertension [120]. Additionally, once it reaches its target protease. Peptide sequences, cathepsins have been found to be relevant biomarkers which are stable in the blood but highly specific to- in various diseases beyond cancer, like lung and brain wards target proteases, have been developed. To fa- diseases and atherosclerosis [121]. Further reading on cilitate delivery of therapeutic agents to the target site, the use of proteases as biomarkers and targets in dis- targeting moieties (e.g. antibody) or macromolecular eases outside of cancer can be found in reference [3] carriers (e.g. albumin or polymers) are coupled to the and references within. peptide-based prodrugs. Therefore, in most cases, Proteases are important regulatory molecules in prodrugs are composed of two major components: i) our body. Their proper function is needed for appro- an active therapeutic agent and ii) peptide substrates priate protein turnover and signaling. However, and/or macromolecular carriers, i.e. promoiety. To when these proteases are unbalanced their irreversi- endow prodrugs with target-specificity, they can also ble actions can greatly affect normal cellular function be modified with additional iii) targeting ligands [12, and lead to pathologies like cancer and neurodegen- 123]. In other words, the parent therapeutic agent is erative, pulmonary and cardiovascular diseases. caged by the promoiety and/or modified with the Many studies have suggested the use of different targeting ligand via target-specific, cleavable linkages proteases as biomarkers and prognostic markers for (Fig. 1). Prodrugs activated by proteolysis have at- such diseases. Furthermore, protease inhibitors have tracted much attention in the field of drug discovery been developed to stabilize protease function. Here and development. In this section, representative PAP we briefly introduced key human proteases that have approaches including peptide-based prodrugs and been found to be overexpressed in various patholo- macromolecular prodrugs will be briefly exemplified. gies, specifically cancer. Furthermore, we describe how they are deregulated and list the proteases 3.1. Cathepsin-Activatable Prodrugs commonly implicated in various forms of disorders (Table 1). We hope that this protease introduction can Proteases are known to selectively cleave specific serve to educate readers on potential drug targets and substrates, e.g. an amino acid or peptide sequences. how their overexpression can be utilized for specific The fact that proteases can recognize and degrade drug delivery by PAPs. specific substrates sheds light on the development of prodrugs that can be activated at target disease sites. 3. Protease-Activatable Prodrugs Among proteases, cathepsins are well-known prote- ases that are upregulated in several tumor tissues Prodrugs are chemically-caged derivatives of (Table 1). Overexpressed cathepsins are considered therapeutic agents that can be transformed by an en- important biomarkers for cancer and can serve as key zyme, chemical or physiological stimuli to release the targets for prodrugs to induce anticancer drug release active parent drug in vivo [12]. From the time the term to tumor tissues or inside tumor cells. prodrug was first coined by Adrien Albert in 1958 Initial works on protease prodrugs focused on [122], a wide variety of prodrugs has been extensively conjugating single amino acids or dipeptides onto investigated. The prodrug approach generally aims to common cancer therapeutic drugs like daunorubicin i) improve the physicochemical properties of parent (DNR) or doxorubicin (DOX) to study their increased drug molecules, such as low chemical stability or poor therapeutic effects. water-solubility; ii) improve their pharmacokinetic/ pharmacologic properties, like insufficient oral ab-

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Fig. 1 Schematic diagram of (A-E) prodrug constructs and (F) hypothetical pathway of prodrug activation

For example, amino acid- and dipep- and lung carcinoma models. Notably, it was sug- tide-daunorubicin conjugates (L-Leu-DNR, Val-DNR, gested that superior antitumor efficacy of Leu-DOX to Ile-DNR, Ala-Leu-DNR and Leu-Leu-DNR) were DOX is due to their enhanced hydrophobicity and prepared to investigate the effect of modification of also to their proteolysis in tumor tissue by proteases DNR on its toxicity and antitumor efficacy toward such as cathepsins, which are known to be highly ex- murine L1210 leukemia [124]. When intravenously pressed in several tumor tissues (Table 1). However, administrated into L1210 leukemia xenografts, the studies did not show any direct evidence of pro- Leu-DNR, Ala-Leu-DNR and Leu-Leu-DNR exhibited teolysis of the leucyl group by cathepsins. superior results on the suppression of tumor growth Follow up studies investigated the peptide sub- to DNR and on the survival rate. Leu-DNR accumu- strates, proteolysis mechanisms and prodrug delivery lated in the heart muscle much less than DNR at eq- agents like albumin and macromolecules on cancer uitoxic doses in the rabbit, and therefore exhibited therapeutic effects. DNR or DNR derivatives (DNR, reduced cardiotoxicity, a major side effect of an- Leu-DNR, Ala-Leu-DNR, Leu-Ala-Leu-DNR or thracycline derivatives. Similarly, Leu-DOX prodrugs Ala-Leu-Ala-Leu-DNR) were further conjugated onto were also developed to lower the cardiotoxicity and albumin (ALB) [131]. Remarkably, when the DNR improve the therapeutic index of DOX [125-130]. prodrugs were incubated with lysosomal enzymes, Compared to DOX alone, Leu-DOX had higher anti- active DNR molecules were released from tumor efficacy together with lower toxicity in various ALB-Leu-Ala-Leu-DNR and ALB-Ala-Leu-Ala-Leu- tumor-bearing mice including human ovarian, breast DNR but not from ALB-DNR, ALB-Leu-DNR or

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ALB-Ala-Leu-DNR. However, in the presence of 95% period of time, the so called enhanced permeable and calf serum, ALB-Leu-Ala-Leu-DNR and retention (EPR) effect [132-133]. In an effort to im- ALB-Ala-Leu-Ala-Leu-DNR were over 97% stable prove blood stability and tumor targetability of pro- after 24 h incubation. Likewise, in the intraperitoneal drugs, macromolecule-based prodrugs have been L1210 leukemia model, the ALB-tri-/tetra-peptide- developed. As an example, N-(2-hydroxypropyl) DNR prodrugs were proven to be superior to methacrylamide copolymer (HPMAcp) was exploited mono-peptide linkers, with improved anticancer ac- as a macromolecular carrier to deliver therapeutic tivity and prolonged survivals. From the results, it agents or their derivatives. Onto the HPMAcp back- was proposed that the ALB-tri-/tetra-peptide-DNR bone, DOX was conjugated via a cathepsin B-specific prodrugs internalize into leukemia cells and release tetrapeptide (Gly-Phe-Leu-Gly) linker [134-140]. After parent DNR molecules inside the cells because of intravenous administration, the macromolecular proteolysis of the peptide substrates by lysosomal prodrug circulated approximately 15-times longer proteases, like cathepsin B. than free DOX. Furthermore, the prodrug exhibited Besides albumin, macromolecules have been 100-times less heart uptake, which resulted in de- vigorously investigated as potent drug-delivery car- creased toxicity and improved therapeutic efficacy riers. Owing to the abnormal characteristics of tumors compared with free DOX. Interestingly, the pharma- like leaky blood vessels and lack of lymphatic drain- cokinetics and amount of released DOX was found to age, macromolecules are known to preferentially ac- be correlated with both lysosomal activity of cathep- cumulate in tumor tissue and remain for a prolonged sin B and vascular properties (Fig. 2) [135].

Fig. 2 (A) Chemical structure of PK1 (HPMAcp-Gly-Phe-Leu-Gly-DOX). DOX concentrations in MAC15A (◯) and MAC26 (□) plasma (dotted line) and tumor (solid line) after administration of 10 mg/kg DOX i.v (B) or 490 mg/kg PK1, i.v. (40 mg/kg DOX equivalent) (C). Antitumor effects of DOX or PK1 against MAC26 tumors (D), MAC15A (E and F). ◯, PK1 (40 mg/kg); □, PK1 (20 mg/kg); △, PK1 (10 mg/kg); ▽, dox (10 Mg/kg); ◇, controls. Adapted with permission from Loadman et al [135]. Copyright 1999, American Association for Cancer Research

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This study implies that highly-activated cathep- ase of interest in found intracellularly. In the above sin B can induce vascularization around tumors and examples, the use of macromolecular substrates or the accelerate DOX release. Most importantly, the study addition of albumin greatly increased the delivery of exemplifies that a protease can enhance the therapeu- the prodrug to its target site. tic efficacy of a macromolecular prodrug. However, the study could not elucidate whether the excellent 3.2. PSA-activatable prodrugs therapeutic efficacy was attributed to DOX release activated by cathepsin B or a higher accumulation of The peptide sequence (Ser-Ser-Lys-Leu-Gln) is the prodrug through highly vascularized tumor ves- highly specific for PSA [147]. The peptide substrate sels. With the apparent enhanced drug efficacy, has been coupled with DOX to develop a HPMAcp was also conjugated to DNR [141], PSA-activatable prodrug [148-149]. The resulting 5-fluorouracil (5-FU)[142], platinum (II) [143] and prodrug (morpholinocarbonyl(Mu)-Ser-Ser-Lys-Leu- O-(chloracetyl-carbamoyl) fumagillol (TNP-470) [144] Gln-DOX) exerted severe cytotoxicity not only to via the same peptide linker (Gly-Phe-Leu-Gly). Just as PC-82 human prostate cancer cells, that secrete high the DOX conjugate, these formulations also showed levels of PSA, but also to LNCaP human prostate significant therapeutic activity to free drugs. cancer cells, that secrete 30 fold less active PSA. Yet A significant example of cathepsin B-activatable following 72-h incubation with LNCaP cells, over 90% prodrugs utilized a polymerized substrate to reverse of the prodrug was activated to release Leu-DOX and cancer progression, emphasizing the use of macro- killed 50% of cells at a low concentration (IC50 = 70 molecular protease substrates for efficient drug de- nM). Importantly, the drug did not show detectable livery and effective therapy. The cathepsin toxicity to PSA-nonproducing cells, TSU human B-activatable prodrug, poly(L-glutamic acid) conju- prostate cancer cells, which indicates great sensitivity gated with paclitaxel (PTX), exerted impressive anti- of the PSA-activatable prodrug. When the prodrug tumor activity in OCA-1 ovarian carcinoma models was given by continuous infusion into PC-82 human [145]. Notably, a single intravenous injection of the prostate cancer xenografts at a high dose (four times macromolecular prodrug resulted in the disappear- higher than the 100% lethal dose of free DOX), no ance of 13762F adenocarcinoma implanted in rats and noticeable toxicity was observed. Moreover, an in- OCA-1 ovarian carcinoma inoculated in rats. The ex- traperitoneal injection of the prodrug suppressed cellent antitumor activity possibly resulted from its tumor growth up to 57% in tumor weight compared prolonged half-life in plasma and preferential accu- with the non-treated control group. The continuous mulation of released PTX in tumor tissue. In a fol- infusion with the prodrug also led to similar sup- low-up study, the metabolic pathway of the macro- pression activity on tumor growth (47% lower than molecular prodrug, poly(L-glutamic acid)-PTX was control group in tumor weight) at 60% lower dose investigated [146]. The study demonstrated that it can than the dose administrated intraperitoneally. In the be metabolized into mono- or di-glutamyl-PTX form same way, thapsigargin (TPG) [150-151] or after it internalizes into cancer cells. The release rate of 5-fluorodeoxyuridine (5-FudR) [152] were coupled PTX is correlated with the proteolytic activity of ca- with the same PSA-specific peptide substrate. The thepsin B. The metabolism of poly(L-glutamic ac- PSA-activatable prodrugs also exhibited remarkable id)-PTX was inhibited in cathepsin B deficient mice or inhibitory effects on tumor growth without discerni- by administration of cathepsin B inhibitor in mice. ble toxicity after intravenous administration. Although cathepsin B is a common protease Another peptide substrate (N-glutaryl- target for prodrugs (Table 2), its proteolytic activity (4-hydroxyprolyl)-Ala-Ser-chGly-Gln-Ser-Leu) spe- needs to be examined more closely. For example, cific for PSA was also discovered and exploited for the previous works have shown that cathepsin B can be development of PSA-activatable prodrugs (Fig. 3) released to the extracellular space in active form un- [153]. In this research, DOX was covalently incorpo- der certain pathologies (Section 3.1). This may cause rated with PSA-specific peptide sequence. The re- premature activity of the prodrug and reduce its effi- sulting conjugate was hydrolyzed specifically by PSA cacy inside tumor cells. However, cathepsin activata- and transformed into cytotoxic form of DOX, leu- ble prodrugs are exemplary in their roles for thera- cine-DOX and DOX. Like other prodrugs, it was much peutic delivery because these PAPs are shown to be less toxic than free DOX to PSA-negative cells; how- much more efficient than equitoxic doses of free ever, it led to remarkable reduction in tumor burden drugs. It is important to consider additional molecules in human prostate cancer xenografts inoculated with for proper drug delivery, especially when the prote- either LNCaP or CWR22.

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Fig. 3 (A) Chemical structure of L-377,202. Changes in concentrations of leucine–DOX and DOX in tumor tissue (B) and heart tissue (C). ◆, DOX from L-377,202; ●, leucine–DOX from L-377,202; ◇, DOX from administration of conventional DOX. (D) Reduction in LNCaP tumor weights in nude mice treated with DOX or L-377,202. Adapted with permission from DeFeo-Jones et al [153]. Copyright 2000, Nature Publishing Group

Macromolecules, such as ALB and HPMAcp, used as a linker to conjugate TPG derivative named were also combined with therapeutic agents like TPG L12ADT onto the HPMAcp backbone [154]. In an- or DOX via PSA-specific substrates. In a macromo- other system, DOX was coupled with ALB via a sim- lecular prodrug system, the same peptide sequence ilar peptide substrate (ε-maleimidocaproic acid previously described (Mu-Ser-Ser-Lys-Leu-Gln), was (EMC)-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Gly or -Arg)

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[155-156]. PSA selectively cleaves the substrate, con- Just as tumor-specific chemotherapeutics, verting inactive prodrugs into active forms of parent MMP-activatable prodrugs can be improved by uti- drugs or their derivatives. As an example, lizing a macromolecular carrier. For example, meto- ALB-EMC-Arg-Arg-Ser-Ser-Tyr-Tyr-Ser-Arg-DOX traxate (MTX) was conjugated onto a macromolecular exerted enhanced suppression of tumor growth by carrier, dextran (DEX) via an MMP-specific peptide 62% in tumor size over free DOX. Additionally, the linker (Pro-Val-Gly-Leu-Ile-Gly) (Fig. 4) [160-161]. prodrug showed no sign of detectable toxicity after This macromolecular prodrug shows specificity to administration of equimolar DOX into a prostate MMP-2 and -9 and releases peptidyl-MTX when in- cancer model orthotopically implanted with LNCaP cubated with targeted proteases; however, it re- LN prostate cancer cells. More interestingly, admin- mained stable in serum. This study illustrated that istration of the prodrug reduced the metastatic bur- macromolecular prodrugs preferentially accumulate den in the lungs by 50%. in tumor tissues by the EPR effect more than free MTX 3.3. MMP-activatable prodrugs and then release MTX derivatives close to or within tumor tissues. Importantly, the amount of tumor ac- MMPs are representative target protease for cumulation in vivo was not significantly correlated prodrugs that may arguably be the most studied with the specificity of peptide substrates, even though proteases. Because they are found in the extracellular the peptide sequence was proven to be highly specific and pericellular areas of the cell, MMPs can serve as in vitro. The unexpected results suggest that since excellent protease targets for prodrug delivery to the there is a great number of endo- and in tumor microenvironment. Although a versatile fami- the human body, the in vivo properties, such as stabil- ly, specific substrates have been discovered that can ity in the bloodstream and the specificity to the target be cleaved by a limited amount of MMPs. In particu- protease, should be carefully considered. lar, the progression of various malignancies can be MMP overexpression is linked to numerous characterized by the overexpression of MMP-2 [2]. cancers (Table 1). Their role in ECM remodeling is Like the other prodrugs described above, required for cancer cells to metastasize and invade MMP-activatable prodrugs have been developed by different locations. Therefore MMP-specific prodrugs chemical conjugation of MMP-cleavable peptide sub- can especially target metastasized forms of cancer. A strate (Glu-Pro-Cit-Gly-Hof-Tyr-Leu) to DOX drawback to MMP targeting is their cleavage of vari- [157-158]. The peptide substrate was specifically ous substrates by multiple types of MMPs. The ex- cleaved by MMP-2, -9, -14. Interestingly, it was found emplified prodrugs in this section utilized unique that the peptide substrate was initially metabolized by peptide sequences but still targeted more than one neprilysin, a membrane-bound metalloproteinase, MMP. Although this may be a limitation for molecu- when they were incubated with an MMP-expressing lar biology in order to identify a specific form of fibrosarcoma cell line, HT1080. In HT1080 xenografts, MMP, multiple-MMP targeted prodrugs can be used the substrate was preferentially metabolized in the to deliver anticancer drugs to especially harmful tumor tissue, resulting in a 10-fold higher accumula- forms of cancer. tion of released DOX at the tumor than that in the 3.4. Targeted protease-activatable prodrugs heart. When this MMP-substrate prodrug was ad- ministered to mice, 80% of mice were cured and no As we reviewed in the previous sections, PAPs significant toxicity was detected, such as weight loss are generally inactive but become pharmacologically or marrow toxicity. active when exposed to target proteases. Owing to its Since MMP-2, -9 specifically cleave the peptide improved stability and target-specific drug release, sequence Gly-Pro-Leu-Gly-Ile-Ala-Gly-Gln, the pep- prodrugs have a wide therapeutic window when tide substrate was also used to design a DOX prodrug compared to free chemotherapeutic agents. However, that can be specifically activated by MMP-2 [159]. in terms of the targeting mechanism, most conven- ALB was incorporated with the peptide-DOX conju- tional prodrugs are still limited because they mainly gate as a carrier. The intact albumin-peptide-DOX rely on passive accumulation pathways, e.g. EPR ef- conjugate was not significantly toxic. However, fol- fect. In an effort to bestow disease-specific targetabil- lowing addition of MMP-2, the albumin-peptide-DOX ity upon prodrugs, various targeting moieties, e.g. conjugate released tetrapeptide-DOX and was further antibody, peptide or oligonucleic acid molecules, transformed into the parent anticancer drug, DOX. have been employed. The prodrug was 4-fold less toxic than free DOX in For instance, therapeutic agents have been co- vivo, whereas it showed superior anticancer effect to valently conjugated with disease-specific antibodies DOX at an equitoxic dose. via protease-cleavable linkages to incorporate both a

http://www.thno.org Theranostics 2012, 2(2) 169 tumor-specific recognition site and a tumor selective plasma but released active MMAE in lysosomes of enzymatic activation sequence in a single prodrug CD30-positive tumor cells. The ADC showed higher platform. The antibody-drug conjugates (ADCs) have specificity in vitro, lower toxicity in vivo and improved come a long way and shown significant therapeutic antitumor effect than control ADCs containing hy- efficacy against various diseases [162-163]. An inter- drazone linker. Notably, with the use of this ADC, the esting study demonstrates the development and in therapeutic index significantly increased up to 60-fold vivo applications of a cathepsin activated ADC. A leading to its extensive investigation in the clinic. Re- synthetic dolastatin 10 analog, monomethyl auristatin cently, the conjugate, named Adcetris (brentuximab E (MMAE), was incorporated onto an antibody cAC10 vedotin) has been approved by The U.S. Food and with specificity for CD30 on hematological malignan- Drug Administration (FDA) for the treatment of cies via a dipeptide substrate (Val-Cit) as a cathepsin Hodgkin lymphoma (HL) and a rare lymphoma B-specific linker (Fig. 5) [164]. The known as systemic anaplastic large cell lymphoma mAb-Val-Cit-MMAE conjugate is highly stable in (ALCL). [165]

Fig. 4 (A) Chemical structure of the MTX-PVGLIG-DEX. (B) Cytotoxicity of MTX and different types of MTX-peptide analogs: MTX (△), MTX-G (□), MTX-GI (◯), MTX-GIV (▲), and MTXPVG (■). (C) Stability of the conjugate (MTX-PVGLIG-DEX) in various media con- ditions. Tissue distribution of MTX equivalent in mice receiving MTX-PVGLIG-DEX (D) and free MTX (E), at 5 h (▩) and 24 h (■) post injection. Adapted with permission from Chau et al [160-161]. Copyright 2004, American Chemical Society and Copyright 2006, John Wiley & Sons, Inc

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Fig. 5 (A) Chemical structure of mAb-drug conjugates. (B) Hydrolysis of cBR96-Phe-Lys-MMAE and cBR96-Val-Cit-MMAE (eight drug-mAb combinations) with human cathepsin B. In vivo therapeutic efficacy of the conjugates in immunocompromised mice with sub- cutaneous human tumor xenografts. (C) Athymic mice with subcutaneous L2987 human lung adenocarcinoma tumors (cBR96 Ag+, cAC10 Ag–) were treated with the conjugates. (D) SCID mice with subcutaneous Karpas 299 human ALCL tumors (cAC10 Ag+, cBR96 Ag–) were treated with MMAE or with the mAb-Val-Cit-MMAE. (E) SCID mice with Karpas 299 tumors were treated with cAC10, cAC10 + unconjugated MMAE, cAC10-Val-Cit-MMAE or cBR96-Val-Cit-MMAE. Adapted with permission from Doronina et al [164]. Copyright 2003, Nature Publishing Group

Another example of targeted PAPs is macromo- (GAL) to facilitate liver targeting, lecular prodrugs, PK1 and PK2. As we described, GAL-HPMAcp-Gly-Phe-Leu-Gly-DOX. After intra- HPMAcp-Gly-Phe-Leu-Gly-DOX, called PK1, is a venous injection of PK2, acute toxicity decreased three macromolecular prodrug targeting cathepsin B times over free DOX without noticeable signs of car- [134-135, 137-140]. When compared with free DOX, diotoxicity. With these promising results, PK1 and PK1 showed prolonged circulation, less cardiotoxicity PK2 are now under clinical trials [166-171]. and improved therapeutic activity. In addition, it was In addition to mAb or GAL, a bicyclic peptide proposed that the excellent therapeutic efficacy of sequence, RGD-4C (Cys-Asp-Cys-Arg-Gly-Asp- PK1 is due to the passive accumulation into the tumor Cys-Phe-Cys) was exploited as a targeting moiety tissue by the EPR effect and the target-specific drug [172]. RGD-4C selectively binds αvβ3 and αvβ5 integ- release by lysosomal degradation of the peptide rins, which are known to highly overexpress on in- linkage. PK2 was further modified with galactose vading tumor endothelial cells. The targeting peptide

http://www.thno.org Theranostics 2012, 2(2) 171 was coupled with DOX via a plasmin-specific peptide ase overexpressed environments. We acknowledge substrate (D-Ala-Phe-Lys). As we mentioned, plasmin many proteases require further research in their is highly associated with tumor progression (Section physiological roles, but the overwhelming research 2.2). The resulting conjugate exerted cytotoxicity showing their overexpression in pathologies cannot against plasmin-positive HT1080 fibrosarcoma cells be ignored (Table 1). PAPs do not hamper or inhibit but not to endothelial cells, showing significant speci- normal protease activity directly, but instead use the ficity to the target protease plasmin. Likewise, a cyclic protease deregulation to identify an area that requires 13-mer oligopeptide named pep 42 was exploited as a a specific form of drug. In this way, unnecessary tox- targeting moiety to develop tumor targeted PAPs icity to healthy cells is eliminated. The three design [173]. Pep 42 can bind to glucose-regulated protein elements to consider when developing PAPs are: 1) GRP78 and facilitate cancer cell-specific uptake. An- stability and specificity of peptide substrate, 2) loca- ticancer drug, PTX or DOX was conjugated with pep tion of drug activation, 3) addition of a delivery sys- 42 via a cathepsin B-specific cleavable linker (Val-Cit). tem. The conjugates were designed to localize at lysosomes First, the stability and specificity of a peptide and to release therapeutic agents when taken up by substrate is by far the most important element to effi- the cancer cells. The targeted PAP system exhibited ciently target proteases. The peptide substrate must improved anticancer activity against SJSA-1 osteo- remain stable in blood and plasma until it reaches its sarcoma cells known to express GRP78. target protease upon which, it is cleaved to release the Overall, PAPs have shown superior pharmaco- parent drug. If the peptide is not stable in these con- kinetic properties to free drug molecules because ditions, the prodrug cannot be intravenously admin- chemical modification can improve their chemical istered and does not provide any benefit over admin- stability, hydrophilicity and circulation time in the istration of the parent drug directly. The stability of bloodstream. More importantly, the PAP approach peptide substrates can be enhanced by the use of improves pharmacological properties owing to their macromolecular structures, as seen in the example preferential accumulation and release of active forms above of the polymerized peptide substrate for ca- of therapeutic agents in target tissues or cells by pro- thepsin B. Furthermore, the prodrug should target a teolysis. Additional modification of PAP with target- specific protease that is associated with the disease. ing molecules facilitates the accumulation and release This is an area that requires further research, as many of active therapeutic agents at the target site. Over the specific substrates for proteases linked to cancer have past half-century, a great number of PAPs have been not been identified. For example, MMP substrates invented and used in clinical settings. Based on the target many different MMPs and therefore have low successful examples of the PAP approach, the fol- specificity for one member of the MMP family. Alt- lowing section will elucidate imperative design fac- hough this is not necessarily a disadvantage, it may tors in the development of PAP. still reduce the efficacy of the drug and increase tox- icity to areas that do not require the therapy. Caspa- 4. Perspective on prodrug design ses, on the other hand, have more specific substrates Although a few PAPs have entered clinical de- but they are generally not seen as good targets for velopment already (Table 3), we further identify key cancer because they are key signal molecules for design elements that are needed to develop highly apoptosis, a process normally found in healthy cells efficient therapeutic agents that are released in prote- but circumvented in cancer cells.

Table 3. Protease-activatable prodrugs in the clinic

Name Protease Construct Indication Status Ref. Brentuximab Hodgkin [162- Cathepsin B cAC10-Val-Cit-MMAE Approved vedotin (SGN-35) lymphoma 163] OPAXIO™ [258- Cathepsin B poly-L-glutamic acid-PTX NSCLC Phase III (CT-2103) 260] PK1 Cathepsin B HPMAcp-Gly-Phe-Leu-Gly-DOX Cancer PhaseI/II [170] (FCE 28068) PK2 Cathepsin B GAL-HPMAcp-Gly-Phe-Leu-Gly-DOX Liver cancer PhaseI/II [167] (FCE 28069) L-377,202 Prostate PSA N-glutaryl-(4-hydroxyprolyl)-Ala-Ser- chGly-Gln-Ser-Leu-DOX Phase II [261] (C1853) cancer *Abbreviations: cAC10: chimeric anti-CD30 monoclonal antibody, chGly: cyclohexaglycyl, Cit: citrulline, GAL: galactose, DOX: doxorubicin, HPMAcp: N-(2-hydroxypropyl)methacrylamide copolymer, NSCLC: non-small-cell lung cancer, MMAE: monomethyl auristatin E, PTX: paclitaxel

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Second, location of target protease expression is therapeutic delivery can act as multimodal imaging important to recognize in order to design drug con- and drug delivery agent – a true theranostics agent. jugates that can reach the specific target. In most cas- es, drugs exert their therapeutic effect when delivered 5. Conclusion specifically to the intracellular space of the unhealthy We have introduced key proteases that are in- cell. But certain important target proteases are ex- volved in various pathologies, especially cancer, and pressed extracellularly, especially under pathological their potential as targets for prodrugs. With a brief conditions, and the PAP can be activated prematurely introduction on the roles of cathepsins, kallikreins, outside of the cell. In this view, prodrug constructs serine proteases, caspases and MMPs, we show that including peptide substrates, therapeutic agent and their dysregulation can be implicated in many forms targeting moiety should be designed together with the of cancer, as well as in neurodegenerative, pulmonary target protease and its active site in mind. For exam- and cardiovascular diseases. Furthermore, we pro- ple, PEG improves chemical stability and biocompat- vide few examples of PAPs that have utilized specific ibility of drug molecules and also prolongs circulation proteases to target intracellular cathepsins, PSA (a time when coupled with hydrophobic drug mole- type of human kallikrein), and MMPs. In this way, we cules, but also reduces cellular uptake of drug mole- hope to illuminate the important roles that proteases cules. In that case, the linkage between drug mole- can play in targeted therapies and go beyond their cules and PEG should be cleaved by a specific prote- functional role in molecular imaging. With our expe- ase that is active in extracellular conditions prior to rience in engineering protease activatable probes, we cellular uptake as shown in ref [174]. On the other have identified important design considerations re- hand, if a therapeutic agent works well inside target quired for efficient development of PAPs and further cells but is not stable outside the cells, the drug should imply that such conjugates can play an important role be caged by a specific promoiety that can protect it in theranostics. until it is degraded at target compartments of the cells [164, 175-176]. In such cases, various targeting moie- Acknowledgements ties, like aptamers or antibodies and additional pro- This work was supported by the Intramural Re- moieties may be helpful to target the prodrug into the search Program (IRP) of the National Institutes of intracellular space. Furthermore, function of the pro- Biomedical Imaging and Bioengineering (NIBIB), Na- tease in the type and stage of tumors is significant tional Institutes of Health (NIH). S.L. is partially when designing PAPs. Most enzymes are temporal, supported by the NIH Pathway to Independence for examples caspase 2 and 3 are markers of apoptosis (K99/R00) Award. but caspase 3 is only present at late stages. This may explain why many caspase-based probes have mainly Conflict of Interest served as sensors for apoptosis over drug delivery The authors have declared that no conflict of in- agents. Emerging data, such as for hKs, indicate that terest exists. their function is dependent on hormone balances as well as tissue type. Therefore, it is beneficial to target References enzymes that are present throughout the entire dis- 1. Lopez-Otin C and Hunter T. The regulatory crosstalk between kinases ease progression and to understand the protease role and proteases in cancer. Nat Rev Cancer. 2010; 10: 278-92. in the specific tissue targeted. A solution is to use 2. Lopez-Otin C and Matrisian LM. Emerging roles of proteases in tumour combinatorial delivery approaches as described in the suppression. Nat Rev Cancer. 2007; 7: 800-8. final design consideration. 3. Turk B. Targeting proteases: Successes, failures and future prospects. Nat Rev Drug Discov. 2006; 5: 785-99. Thirdly, a delivery carrier system can be utilized 4. Lopez-Otin C and Bond JS. Proteases: Multifunctional enzymes in life to target the prodrug more efficiently. As seen in the and disease. J Biol Chem. 2008; 283: 30433-7. current prodrug examples, albumin served as a stabi- 5. Orlowski RZ and Kuhn DJ. Proteasome inhibitors in cancer therapy: lizing agent for the therapeutic and also aided in Lessons from the first decade. Clin Cancer Res. 2008; 14: 1649-57. prodrug accumulation by the EPR effect. Nanoparti- 6. Horiguchi A, Zheng R, Goodman OB, Jr., et al. Lentiviral vector neutral gene transfer suppresses prostate cancer tumor growth. cles, as seen in many drug delivery applications, can Cancer Gene Ther. 2007; 14: 583-9. accumulate in the tumor by EPR efficiently [177]. 7. Karikari CA, Roy I, Tryggestad E, et al. Targeting the apoptotic More importantly, nanoparticles can serve as plat- machinery in pancreatic cancers using small-molecule antagonists of the forms for numerous moieties that can aid in PAP de- x-linked inhibitor of apoptosis protein. Mol Cancer Ther. 2007; 6: 957-66. 8. Jang B and Choi Y. Photosensitizer-conjugated gold nanorods for sign like protease substrates, targeting molecules, enzyme-activatable fluorescence imaging and photodynamic therapy. stabilizing agents, and imaging agents. In this way, a Theranostics. 2012; 2: 190-7. nanoparticle based approach for protease targeted

http://www.thno.org Theranostics 2012, 2(2) 173

9. Kim GB and Kim Y-P. Analysis of protease activity using quantum dots. 35. Buck MR, Karustis DG, Day NA, et al. Degradation of Theranostics. 2012; 2: 127-38. extracellular-matrix proteins by human cathepsin b from normal and 10. Yhee JY, Kinm SA, Koo HB, et al. Optical imaging of cancer-related tumour tissues. Biochem J. 1992; 282 ( Pt 1): 273-8. proteases using near-infrared fluorescence matrix 36. Gocheva V, Zeng W, Ke D, et al. Distinct roles for cysteine cathepsin metalloproteinase-sensitive and cathepsin b-sensitive probes. genes in multistage tumorigenesis. Genes Dev. 2006; 20: 543-56. Theranostics. 2012; 2: 179-89. 37. Yan S and Sloane BF. Molecular regulation of human cathepsin b: 11. Zhu L, Xie J, Swierczewska M, et al. Real-time video imaging of protease Implication in pathologies. Biol Chem. 2003; 384: 845-54. expression in vivo. Theranostics. 2011; 1: 18-27. 38. Cavallo-Medved D, Mai J, Dosescu J, et al. Caveolin-1 mediates the 12. Rautio J, Kumpulainen H, Heimbach T, et al. Prodrugs: Design and expression and localization of cathepsin b, pro-urokinase plasminogen clinical applications. Nat Rev Drug Discov. 2008; 7: 255-70. activator and their cell-surface receptors in human colorectal carcinoma 13. Mahato R, Tai W, and Cheng K. Prodrugs for improving tumor cells. J Cell Sci. 2005; 118: 1493-503. targetability and efficiency. Adv Drug Deliv Rev. 2011; 63: 659-70. 39. Boya P and Kroemer G. Lysosomal membrane permeabilization in cell 14. Stella VJ. Prodrugs as therapeutics. Expert Opin Ther Pat. 2004; 14: death. Oncogene. 2008; 27: 6434-51. 277-80. 40. Cardone RA, Casavola V, and Reshkin SJ. The role of disturbed ph 15. Borgono CA and Diamandis EP. The emerging roles of human tissue dynamics and the na+/h+ exchanger in metastasis. Nat Rev Cancer. kallikreins in cancer. Nat Rev Cancer. 2004; 4: 876-90. 2005; 5: 786-95. 16. Gabriel D, Zuluaga MF, Van Den Bergh H, et al. It is all about proteases: 41. Reddy VY, Zhang QY, and Weiss SJ. Pericellular mobilization of the From drug delivery to in vivo imaging and photomedicine. Curr Med tissue-destructive cysteine proteinases, cathepsins b, l, and s, by human Chem. 2011; 18: 1785-805. monocyte-derived macrophages. Proc Natl Acad Sci U S A. 1995; 92: 17. Coussens LM and Werb Z. Inflammation and cancer. Nature. 2002; 420: 3849-53. 860-7. 42. Cirman T, Orešić K, Mazovec GD, et al. Selective disruption of lysosomes 18. Pollard JW. Tumour-educated macrophages promote tumour in hela cells triggers apoptosis mediated by cleavage of bid by multiple progression and metastasis. Nat Rev Cancer. 2004; 4: 71-8. -like lysosomal cathepsins. J Biol Chem. 2004; 279: 3578-87. 19. Mohamed MM and Sloane BF. Cysteine cathepsins: Multifunctional 43. Stoka V, Turk B, Schendel SL, et al. Lysosomal protease pathways to enzymes in cancer. Nat Rev Cancer. 2006; 6: 764-75. apoptosis - cleavage of bid, not pro-caspases, is the most likely route. J 20. Flannery T, Gibson D, Mirakhur M, et al. The clinical significance of Biol Chem. 2001; 276: 3149-57. cathepsin s expression in human astrocytomas. Am J Pathol. 2003; 163: 44. Vasiljeva O and Turk B. Dual contrasting roles of cysteine cathepsins in 175-82. cancer progression: Apoptosis versus tumour invasion. Biochimie. 2008; 21. Mikkelsen T, Yan PS, Ho KL, et al. Immunolocalization of cathepsin b in 90: 380-6. human glioma: Implications for tumor invasion and angiogenesis. J 45. Reinheckel T, Hagemann S, Dollwet-Mack S, et al. The lysosomal Neurosurg. 1995; 83: 285-90. cathepsin l regulates keratinocyte proliferation by 22. Sloane BF, Honn KV, Sadler JG, et al. Cathepsin-b activity in b-16 control of growth factor recycling. J Cell Sci. 2005; 118: 3387-95. melanoma-cells - a possible marker for metastatic potential. Cancer Res. 46. Joyce JA, Baruch A, Chehade K, et al. Cathepsin cysteine proteases are 1982; 42: 980-6. effectors of invasive growth and angiogenesis during multistage 23. Hughes SJ, Glover TW, Zhu X-X, et al. A novel amplicon at 8p22–23 tumorigenesis. Cancer Cell. 2004; 5: 443-53. results in overexpression of cathepsin b in esophageal adenocarcinoma. 47. Conover CA, Perry JE, and Tindall DJ. Endogenous cathepsin Proc Natl Acad Sci U S A. 1998; 95: 12410-5. d-mediated hydrolysis of insulin-like growth factor-binding proteins in 24. Krueger S, Kalinski T, Hundertmark T, et al. Up-regulation of cathepsin cultured human prostatic-carcinoma cells. J Clin Endocrinol Metab. 1995; x in helicobacter pylori gastritis and gastric cancer. J Pathol. 2005; 207: 80: 987-93. 32-42. 48. Tandon AK, Clark GM, Chamness GC, et al. Cathepsin-d and prognosis 25. Campo E, Munoz J, Miquel R, et al. Cathepsin b expression in colorectal in breast-cancer. N Engl J Med. 1990; 322: 297-302. carcinomas correlates with tumor progression and shortened patient 49. Ma YM, Zhao M, Zhong JL, et al. Proteomic profiling of proteins survival. Am J Pathol. 1994; 145: 301-9. associated with lymph node metastasis in colorectal cancer. J Cell 26. Fernández PL, Farré X, Nadal A, et al. Expression of cathepsins b and s in Biochem. 2010; 110: 1512-9. the progression of prostate carcinoma. Int J Cancer. 2001; 95: 51-5. 50. Zaidi N, Hermann C, Herrmann T, et al. Emerging functional roles of 27. Nägler DK, Krüger S, Kellner A, et al. Up-regulation of cathepsin x in cathepsin e. Biochem Biophys Res Commun. 2008; 377: 327-30. prostate cancer and prostatic intraepithelial neoplasia. Prostate. 2004; 60: 51. Matsuo K, Kobayashi I, Tsukuba T, et al. Immunohistochemical 109-19. localization of cathepsins d and e in human gastric cancer: A possible 28. Santamaría I, Velasco G, Cazorla M, et al. Cathepsin l2, a novel human correlation with local invasive and metastatic activities of carcinoma cysteine proteinase produced by breast and colorectal carcinomas. cells. Hum Pathol. 1996; 27: 184-90. Cancer Res. 1998; 58: 1624-30. 52. Tenti P, Romagnoli S, Silini E, et al. Cervical adenocarcinomas express 29. Linnerth NM, Sirbovan K, and Moorehead RA. Use of a transgenic markers common to gastric, intestinal, and pancreatobiliary epithelial mouse model to identify markers of human lung tumors. Int J Cancer. cells. Pathol Res Pract. 1994; 190: 342-9. 2005; 114: 977-82. 53. Uno K, Azuma T, Nakajima M, et al. Clinical significance of cathepsin e 30. Paik S, Shak S, Tang G, et al. A multigene assay to predict recurrence of in pancreatic juice in the diagnosis of pancreatic ductal adenocarcinoma. tamoxifen-treated, node-negative breast cancer. N Engl J Med. 2004; 351: J Gastroenterol Hepatol. 2000; 15: 1333-8. 2817-26. 54. Terris B, Blaveri E, Crnogorac-Jurcevic T, et al. Characterization of gene 31. Bossard MJ, Tomaszek TA, Thompson SK, et al. Proteolytic activity of expression profiles in intraductal papillary-mucinous tumors of the human osteoclast cathepsin k. J Biol Chem. 1996; 271: 12517-24. pancreas. Am J Pathol. 2002; 160: 1745-54. 32. Xia L, Kilb J, Wex H, et al. Localization of rat cathepsin k in osteoclasts 55. Ullmann R, Morbini P, Halbwedl I, et al. Protein expression profiles in and resorption pits: Inhibition of bone resorption and cathepsin adenocarcinomas and squamous cell carcinomas of the lung generated k-activity by peptidyl vinyl sulfones. Biol Chem. 1999; 380: 679-87. using tissue microarrays. J Pathol. 2004; 203: 798-807. 33. Gocheva V and Joyce JA. Cysteine cathepsins and the cutting edge of 56. Cleutjens KBJM, Van Eekelen CCEM, Van Der Korput HaGM, et al. Two cancer invasion. Cell Cycle. 2007; 6: 60-4. androgen response regions cooperate in steroid hormone regulated 34. Palermo C and Joyce JA. Cysteine cathepsin proteases as activity of the prostate-specific antigen promoter. J Biol Chem. 1996; 271: pharmacological targets in cancer. Trends Pharmacol Sci. 2008; 29: 22-8. 6379-88.

http://www.thno.org Theranostics 2012, 2(2) 174

57. Malm J, Hellman J, Hogg P, et al. Enzymatic action of prostate-specific 78. Shi X, Gangadharan B, Brass LF, et al. Protease-activated receptors (par1 antigen (psa or hk3): Substrate specificity and regulation by Zn2+, a and par2) contribute to tumor cell motility and metastasis. Mol Cancer tight-binding inhibitor. Prostate. 2000; 45: 132-9. Res. 2004; 2: 395-402. 58. Yousef GM and Diamandis EP. The new human tissue kallikrein gene 79. Hengartner MO. The biochemistry of apoptosis. Nature. 2000; 407: 770-6. family: Structure, function, and association to disease. Endocr Rev. 2001; 80. Thornberry NA and Lazebnik Y. Caspases: Enemies within. Science. 22: 184-204. 1998; 281: 1312-6. 59. Réhault S, Monget P, Mazerbourg S, et al. Insulin-like growth factor 81. Leist M and Jaattela M. Four deaths and a funeral: From caspases to binding proteins (igfbps) as potential physiological substrates for human alternative mechanisms. Nat Rev Mol Cell Biol. 2001; 2: 589-98. kallikreins hk2 and hk3. Eur J Biochem. 2001; 268: 2960-8. 82. Hunter AM, Lacasse EC, and Korneluk RG. The inhibitors of apoptosis 60. Mikolajczyk SD, Millar LS, Kumar A, et al. Prostatic human kallikrein 2 (iaps) as cancer targets. Apoptosis. 2007; 12: 1543-68. inactivates and complexes with plasminogen activator inhibitor-1. Int J 83. Klein T and Bischoff R. Physiology and pathophysiology of matrix Cancer. 1999; 81: 438-42. metalloproteases. Amino Acids. 2011; 41: 271-90. 61. Takayama TK, Mcmullen BA, Nelson PS, et al. Characterization of hk4 84. Ra H-J and Parks WC. Control of matrix metalloproteinase catalytic (prostase), a prostate-specific serine protease: Activation of the precursor activity. Matrix Biol. 2007; 26: 587-96. of prostate specific antigen (pro-psa) and single-chain urokinase-type 85. Nagase H. Activation mechanisms of matrix metalloproteinases. Biol plasminogen activator and degradation of prostatic acid phosphatase. Chem. 1997; 378: 151-60. Biochemistry. 2001; 40: 15341-8. 86. Page-Mccaw A, Ewald AJ, and Werb Z. Matrix metalloproteinases and 62. Yoshida E, Ohmura S, Sugiki M, et al. Prostate-specific antigen activates the regulation of tissue remodelling. Nat Rev Mol Cell Biol. 2007; 8: single-chain urokinase-type plasminogen activator. Int J Cancer. 1995; 221-33. 63: 863-5. 87. Chakrabarti S and Patel KD. Matrix metalloproteinase-2 (mmp-2) and 63. Pepper MS. Role of the matrix metalloproteinase and plasminogen mmp-9 in pulmonary pathology. Exp Lung Res. 2005; 31: 599-621. activator-plasmin systems in angiogenesis. Arterioscler Thromb Vasc 88. Nguyen M, Arkell J, and Jackson CJ. Human endothelial gelatinases and Biol. 2001; 21: 1104-17. angiogenesis. Int J Biochem Cell Biol. 2001; 33: 960-70. 64. Killian CS, Corral DA, Kawinski E, et al. Mitogenic response of 89. Bergers G, Brekken R, Mcmahon G, et al. Matrix metalloproteinase-9 osteoblast cells to prostate-specific antigen suggests an activation of triggers the angiogenic switch during carcinogenesis. Nat Cell Biol. 2000; latent tgf-β and a proteolytic modulation of cell adhesion receptors. 2: 737-44. Biochem Biophys Res Commun. 1993; 192: 940-7. 90. Van Den Steen PE, Dubois B, Nelissen I, et al. Biochemistry and 65. Fortier AH, Nelson BJ, Grella DK, et al. Antiangiogenic activity of molecular biology of gelatinase b or matrix metalloproteinase-9 prostate-specific antigen. J Natl Cancer Inst. 1999; 91: 1635-40. (mmp-9). Crit Rev Biochem Mol Biol. 2002; 37: 375-536. 66. Desriviéres S, Lu H, Peyri N, et al. Activation of the 92 kda type iv 91. Somiari SB, Somiari RI, Heckman CM, et al. Circulating and by tissue kallikrein. J Cell Physiol. 1993; 157: 587-93. in breast cancer - potential role in classification of patients into 67. Andreasen PA, Egelund R, and Petersen HH. The plasminogen low risk, high risk, benign disease and breast cancer categories. Int J activation system in tumor growth, invasion, and metastasis. Cell Mol Cancer. 2006; 119: 1403-11. Life Sci. 2000; 57: 25-40. 92. Turpeenniemi-Hujanen T. Gelatinases (mmp-2 and-9) and their natural 68. Foekens JA, Peters HA, Look MP, et al. The urokinase system of inhibitors as prognostic indicators in solid cancers. Biochimie. 2005; 87: plasminogen activation and prognosis in 2780 breast cancer patients. 287-97. Cancer Res. 2000; 60: 636-43. 93. Ranuncolo SM, Armanasco E, Cresta C, et al. Plasma mmp-9 (92 69. Pyke C, Kristensen P, Ralfkiaer E, et al. Urokinase-type kda-mmp) activity is useful in the follow-up and in the assessment of plasminogen-activator is expressed in stromal cells and its receptor in prognosis in breast cancer patients. Int J Cancer. 2003; 106: 745-51. cancer-cells at invasive foci in human colon adenocarcinomas. Am J 94. Schmalfeldt B, Prechtel D, Harting K, et al. Increased expression of Pathol. 1991; 138: 1059-67. matrix metalloproteinases (mmp)-2, mmp-9, and the urokinase-type 70. Stephens RW, Nielsen HJ, Christensen IJ, et al. Plasma urokinase plasminogen activator is associated with progression from benign to receptor levels in patients with colorectal cancer: Relationship to advanced ovarian cancer. Clin Cancer Res. 2001; 7: 2396-404. prognosis. J Natl Cancer Inst. 1999; 91: 869-74. 95. Nawrocki B, Polette M, Marchand V, et al. Expression of matrix 71. Duffy MJ, Maguire TM, Mcdermott EW, et al. Urokinase plasminogen metalloproteinases and their inhibitors in human bronchopulmonary activator: A prognostic marker in multiple types of cancer. J Surg Oncol. carcinomas: Quantificative and morphological analyses. Int J Cancer. 1999; 71: 130-5. 1997; 72: 556-64. 72. Miyake H, Hara I, Yamanaka K, et al. Elevation of serum levels of 96. Zucker S and Vacirca J. Role of matrix metalloproteinases (mmps) in urokinase-type plasminogen activator and its receptor is associated with colorectal cancer. Cancer Metastasis Rev. 2004; 23: 101-17. disease progression and prognosis in patients with prostate cancer. 97. Forsyth PA, Wong H, Laing TD, et al. Gelatinase-a (mmp-2), gelatinase-b Prostate. 1999; 39: 123-9. (mmp-9) and membrane type matrix metalloproteinase-1 (mt1-mmp) are 73. Dass K, Ahmad A, Azmi AS, et al. Evolving role of upa/upar system in involved in different aspects of the pathophysiology of malignant human cancers. Cancer Treat Rev. 2008; 34: 122-36. gliomas. Br J Cancer. 1999; 79: 1828-35. 74. Jin E, Fujiwara M, Pan X, et al. Protease-activated receptor (par)-1 and 98. Yong VW, Power C, Forsyth P, et al. Metalloproteinases in biology and par-2 participate in the cell growth of alveolar capillary endothelium in pathology of the nervous system. Nat Rev Neurosci. 2001; 2: 502-11. primary lung adenocarcinomas. Cancer. 2003; 97: 703-13. 99. Ii M, Yamamoto H, Adachi Y, et al. Role of matrix metalloproteinase-7 75. Takeuchi T, Harris JL, Huang W, et al. Cellular localization of (matrilysin) in human cancer invasion, apoptosis, growth, and membrane-type serine protease 1 and identification of angiogenesis. Exp Biol Med. 2006; 231: 20-7. protease-activated receptor-2 and single-chain urokinase-type 100. Wang F, Reierstad S, and Fishman DA. Matrilysin over-expression in plasminogen activator as substrates. J Biol Chem. 2000; 275: 26333-42. mcf-7 cells enhances cellular invasiveness and pro-gelatinase activation. 76. Darmoul D, Gratio V, Devaud H, et al. Protease-activated receptor 2 in Cancer Lett. 2006; 236: 292-301. colon cancer. J Biol Chem. 2004; 279: 20927-34. 101. Coussens LM, Fingleton B, and Matrisian LM. Matrix metalloproteinase 77. Morris DR, Ding Y, Ricks TK, et al. Protease-activated receptor-2 is inhibitors and cancer: Trials and tribulations. Science. 2002; 295: 2387-92. essential for factor viia and xa–induced signaling, migration, and 102. Nagase H, Visse R, and Murphy G. Structure and function of matrix invasion of breast cancer cells. Cancer Res. 2006; 66: 307-14. metalloproteinases and timps. Cardiovasc Res. 2006; 69: 562-73. 103. Nagase H and Woessner JF. Matrix metalloproteinases. J Biol Chem. 1999; 274: 21491-4.

http://www.thno.org Theranostics 2012, 2(2) 175

104. Nelson AR, Fingleton B, Rothenberg ML, et al. Matrix 129. Deprez-De Campeneere D, Baurain R, and Trouet A. Accumulation and metalloproteinases: Biologic activity and clinical implications. J Clin metabolism of new anthracycline derivatives in the heart after iv Oncol. 2000; 18: 1135. injection into mice. Cancer Chemother Pharmacol. 1982; 8: 193-7. 105. Mattson MP. Pathways towards and away from alzheimer's disease. 130. Zbinden G, Decampeenere D, and Baurain R. Preclinical assessment of Nature. 2004; 430: 631-9. the cardiotoxic potential of anthracycline antibiotics: 106. Selkoe DJ and Schenk D. Alzheimer's disease: Molecular understanding N-l-leucyl-doxorubicin. Arch Toxicol Suppl. 1991; 14: 107-17. predicts amyloid-based therapeutics. Annu Rev Pharmacol and Toxicol. 131. Trouet A, Masquelier M, Baurain R, et al. A covalent linkage between 2003; 43: 545-84. daunorubicin and proteins that is stable in serum and reversible by 107. Lichtenthaler SF, Haass C, and Steiner H. Regulated intramembrane lysosomal , as required for a lysosomotropic drug-carrier proteolysis - lessons from amyloid precursor protein processing. J conjugate: In vitro and in vivo studies. Proc Natl Acad Sci U S A. 1982; Neurochem. 2011; 117: 779-96. 79: 626-9. 108. Janus C, Pearson J, Mclaurin J, et al. A[beta] peptide immunization 132. Matsumura Y and Maeda H. A new concept for macromolecular reduces behavioural impairment and plaques in a model of alzheimer's therapeutics in cancer chemotherapy: Mechanism of tumoritropic disease. Nature. 2000; 408: 979-82. accumulation of proteins and the antitumor agent smancs. Cancer Res. 109. Nicoll JaR, Wilkinson D, Holmes C, et al. Neuropathology of human 1986; 46: 6387-92. alzheimer disease after immunization with amyloid-[beta] peptide: A 133. Maeda H and Matsumura Y. Epr effect based drug design and clinical case report. Nat Med. 2003; 9: 448-52. outlook for enhanced cancer chemotherapy. Adv Drug Deliv Rev. 2011; 110. Diamandis EP, Yousef GM, Petraki C, et al. Human kallikrein 6 as a 63: 129-30. biomarker of alzheimer’s disease. Clin Biochem. 2000; 33: 663-7. 134. Duncan R, Coatsworth JK, and Burtles S. Preclinical toxicology of a novel 111. Blennow K, Hampel H, Weiner M, et al. Cerebrospinal fluid and plasma polymeric antitumour agent: Hpma copolymer-doxorubicin (pk1). Hum biomarkers in alzheimer disease. Nat Rev Neurol. 2010; 6: 131-44. Exp Toxicol. 1998; 17: 93-104. 112. Edwards DR, Handsley MM, and Pennington CJ. The 135. Loadman PM, Bibby MC, Double JA, et al. Pharmacokinetics of pk1 and metalloproteinases. Mol Asp Med. 2008; 29: 258-89. doxorubicin in experimental colon tumor models with differing 113. Montaner J, Molina CA, Monasterio J, et al. Matrix metalloproteinase-9 responses to pk1. Clin Cancer Res. 1999; 5: 3682-8. pretreatment level predicts intracranial hemorrhagic complications after 136. Malugin A, Kopeckova P, and Kopecek J. Liberation of doxorubicin from thrombolysis in human stroke. Circulation. 2003; 107: 598-603. hpma copolymer conjugate is essential for the induction of cell cycle 114. Romanic AM, White RF, Arleth AJ, et al. Matrix metalloproteinase arrest and nuclear fragmentation in ovarian carcinoma cells. J Control expression increases after cerebral focal ischemia in rats - inhibition of Release. 2007; 124: 6-10. matrix metalloproteinase-9 reduces infarct size. Stroke. 1998; 29: 1020-30. 137. Minko T, Kopeckova P, and Kopecek J. Comparison of the anticancer 115. Zhang KY, Mcquibban GA, Silva C, et al. Hiv-induced metalloproteinase effect of free and hpma copolymer-bound adriamycin in human ovarian processing of the chemokine stromal cell derived factor-1 causes carcinoma cells. Pharm Res. 1999; 16: 986-96. neurodegeneration. Nat Neurosci. 2003; 6: 1064-71. 138. Minko T, Kopeckova P, Pozharov V, et al. Hpma copolymer bound 116. Lee CG, Homer R, Zhou Z, et al. Interleukin-13 induces tissue fibrosis by adriamycin overcomes mdr1 gene encoded resistance in a human selectively stimulating and activating transforming growth factor ovarian carcinoma cell line. J Control Release. 1998; 54: 223-33. beta(1). J Exp Med. 2001; 194: 809-21. 139. Seymour LW, Ulbrich K, Strohalm J, et al. The pharmacokinetics of 117. Spinale FG. Myocardial matrix remodeling and the matrix polymer-bound adriamycin. Biochem Pharmacol. 1990; 39: 1125-31. metalloproteinases: Influence on cardiac form and function. Physiol Rev. 140. Yeung TK, Hopewell JW, Simmonds RH, et al. Reduced cardiotoxicity of 2007; 87: 1285-342. doxorubicin given in the form of n-(2-hydroxypropyl)methacrylamide 118. Zaman MA, Oparil S, and Calhoun DA. Drugs targeting the conjugates: And experimental study in the rat. Cancer Chemother renin-angiotensin-aldosterone system. Nat Rev Drug Discov. 2002; 1: Pharmacol. 1991; 29: 105-11. 621-36. 141. Duncan R, Kopeckova P, Strohalm J, et al. Anticancer agents coupled to 119. Jessup M and Brozena S. Heart failure. N Engl J Med. 2003; 348: 2007-18. n-(2-hydroxypropyl)methacrylamide copolymers. Ii. Evaluation of 120. Holland OB, Chud JM, and Braunstein H. Urinary kallikrein excretion in daunomycin conjugates in vivo against l1210 leukaemia. Br J Cancer. essential and mineralocorticoid hypertension. J Clin Invest. 1980; 65: 1988; 57: 147-56. 347-56. 142. Putnam D and Kopecek J. Enantioselective release of 5-fluorouracil from 121. Berdowska I. Cysteine proteases as disease markers. Clin Chim Acta. n-(2-hydroxypropyl)methacrylamide-based copolymers via lysosomal 2004; 342: 41-69. enzymes. Bioconjug Chem. 1995; 6: 483-92. 122. Albert A. Chemical aspects of selective toxicity. Nature. 1958; 182: 421-2. 143. Gianasi E, Buckley RG, Latigo J, et al. Hpma copolymers platinates 123. Law B and Tung CH. Proteolysis: A biological process adapted in drug containing dicarboxylato ligands. Preparation, characterisation and in delivery, therapy, and imaging. Bioconjug Chem. 2009; 20: 1683-95. vitro and in vivo evaluation. J Drug Target. 2002; 10: 549-56. 124. Baurain R, Masquelier M, Deprez-De Campeneere D, et al. Amino acid 144. Satchi-Fainaro R, Puder M, Davies JW, et al. Targeting angiogenesis with and dipeptide derivatives of daunorubicin. 2. Cellular pharmacology a conjugate of hpma copolymer and tnp-470. Nat Med. 2004; 10: 255-61. and antitumor activity on l1210 leukemic cells in vitro and in vivo. J Med 145. Li C, Yu DF, Newman RA, et al. Complete regression of well-established Chem. 1980; 23: 1171-4. tumors using a novel water-soluble poly(l-glutamic acid)-paclitaxel 125. Boven E, Hendriks HR, Erkelens CA, et al. The anti-tumour effects of the conjugate. Cancer Res. 1998; 58: 2404-9. prodrugs n-l-leucyl-doxorubicin and vinblastine-isoleucinate in human 146. Shaffer SA, Baker-Lee C, Kennedy J, et al. In vitro and in vivo ovarian cancer xenografts. Br J Cancer. 1992; 66: 1044-7. metabolism of paclitaxel poliglumex: Identification of metabolites and 126. Breistol K, Hendriks HR, Berger DP, et al. The antitumour activity of the active proteases. Cancer Chemother Pharmacol. 2007; 59: 537-48. prodrug n-l-leucyl-doxorubicin and its parent compound doxorubicin in 147. Denmeade SR, Lou W, Lovgren J, et al. Specific and efficient peptide human tumour xenografts. Eur J Cancer. 1998; 34: 1602-6. substrates for assaying the proteolytic activity of prostate-specific 127. Breistol K, Hendriks HR, and Fodstad O. Superior therapeutic efficacy of antigen. Cancer Res. 1997; 57: 4924-30. n-l-leucyl-doxorubicin versus doxorubicin in human melanoma 148. Denmeade SR, Nagy A, Gao J, et al. Enzymatic activation of a xenografts correlates with higher tumour concentrations of free drug. doxorubicin-peptide prodrug by prostate-specific antigen. Cancer Res. Eur J Cancer. 1999; 35: 1143-9. 1998; 58: 2537-40. 128. De Jong J, Klein I, Bast A, et al. Analysis and pharmacokinetics of 149. Khan SR and Denmeade SR. In vivo activity of a psa-activated n-l-leucyldoxorubucin and metabolites in tissues of tumor-bearing doxorubicin prodrug against psa-producing human prostate cancer balb/c mice. Cancer Chemother Pharmacol. 1992; 31: 156-60. xenografts. Prostate. 2000; 45: 80-3.

http://www.thno.org Theranostics 2012, 2(2) 176

150. Denmeade SR, Jakobsen CM, Janssen S, et al. Prostate-specific 169. Greco F and Vicent MJ. Combination therapy: Opportunities and antigen-activated thapsigargin prodrug as targeted therapy for prostate challenges for polymer-drug conjugates as anticancer nanomedicines. cancer. J Natl Cancer Inst. 2003; 95: 990-1000. Adv Drug Deliv Rev. 2009; 61: 1203-13. 151. Jakobsen CM, Denmeade SR, Isaacs JT, et al. Design, synthesis, and 170. Seymour LW, Ferry DR, Kerr DJ, et al. Phase II studies of pharmacological evaluation of thapsigargin analogues for targeting polymer-doxorubicin (pk1, fce28068) in the treatment of breast, lung and apoptosis to prostatic cancer cells. J Med Chem. 2001; 44: 4696-703. colorectal cancer. Int J Oncol. 2009; 34: 1629-36. 152. Mhaka A, Denmeade SR, Yao W, et al. A 5-fluorodeoxyuridine prodrug 171. Duncan R and Vicent MJ. Do hpma copolymer conjugates have a future as targeted therapy for prostate cancer. Bioorg Med Chem Lett. 2002; 12: as clinically useful nanomedicines? A critical overview of current status 2459-61. and future opportunities. Adv Drug Deliv Rev. 2010; 62: 272-82. 153. Defeo-Jones D, Garsky VM, Wong BK, et al. A peptide-doxorubicin 172. De Groot FM, Broxterman HJ, Adams HP, et al. Design, synthesis, and 'prodrug' activated by prostate-specific antigen selectively kills prostate biological evaluation of a dual tumor-specific motive containing tumor cells positive for prostate-specific antigen in vivo. Nat Med. 2000; integrin-targeted plasmin-cleavable doxorubicin prodrug. Mol Cancer 6: 1248-52. Ther. 2002; 1: 901-11. 154. Chandran SS, Nan A, Rosen DM, et al. A prostate-specific antigen 173. Yoneda Y, Steiniger SC, Capkova K, et al. A cell-penetrating peptidic activated n-(2-hydroxypropyl) methacrylamide copolymer prodrug as grp78 ligand for tumor cell-specific prodrug therapy. Bioorg Med Chem dual-targeted therapy for prostate cancer. Mol Cancer Ther. 2007; 6: Lett. 2008; 18: 1632-6. 2928-37. 174. Kawakami S, Munakata C, Fumoto S, et al. Novel galactosylated 155. Kratz F, Mansour A, Soltau J, et al. Development of albumin-binding liposomes for hepatocyte-selective targeting of lipophilic drugs. J Pharm doxorubicin prodrugs that are cleaved by prostate-specific antigen. Arch Sci. 2001; 90: 105-13. Pharm (Weinheim). 2005; 338: 462-72. 175. Sutherland MS, Sanderson RJ, Gordon KA, et al. Lysosomal trafficking 156. Graeser R, Chung DE, Esser N, et al. Synthesis and biological evaluation and cysteine protease metabolism confer target-specific cytotoxicity by of an albumin-binding prodrug of doxorubicin that is cleaved by peptide-linked anti-cd30-auristatin conjugates. J Biol Chem. 2006; 281: prostate-specific antigen (psa) in a psa-positive orthotopic prostate 10540-7. carcinoma model (lncap). Int J Cancer. 2008; 122: 1145-54. 176. Erickson HK, Park PU, Widdison WC, et al. Antibody-maytansinoid 157. Albright CF, Graciani N, Han W, et al. Matrix conjugates are activated in targeted cancer cells by lysosomal metalloproteinase-activated doxorubicin prodrugs inhibit ht1080 degradation and linker-dependent intracellular processing. Cancer Res. xenograft growth better than doxorubicin with less toxicity. Mol Cancer 2006; 66: 4426-33. Ther. 2005; 4: 751-60. 177. Brigger I, Dubernet C, and Couvreur P. Nanoparticles in cancer therapy 158. Kline T, Torgov MY, Mendelsohn BA, et al. Novel antitumor prodrugs and diagnosis. Adv Drug Deliv Rev. 2002; 54: 631-51. designed for activation by matrix metalloproteinases-2 and -9. Mol 178. Littlewood-Evans AJ, Bilbe G, Bowler WB, et al. The Pharm. 2004; 1: 9-22. osteoclast-associated protease cathepsin k is expressed in human breast 159. Mansour AM, Drevs J, Esser N, et al. A new approach for the treatment carcinoma. Cancer Res. 1997; 57: 5386-90. of malignant melanoma: Enhanced antitumor efficacy of an 179. Liu JA, Sukhova GK, Sun JS, et al. Lysosomal cysteine proteases in albumin-binding doxorubicin prodrug that is cleaved by matrix atherosclerosis. Arterioscler Thromb Vasc Biol. 2004; 24: 1359-66. metalloproteinase 2. Cancer Res. 2003; 63: 4062-6. 180. Sukhova GK, Shi GP, Simon DI, et al. Expression of the elastolytic 160. Chau Y, Tan FE, and Langer R. Synthesis and characterization of cathepsins s and k in human atheroma and regulation of their dextran-peptide-methotrexate conjugates for tumor targeting via production in smooth muscle cells. J Clin Invest. 1998; 102: 576-83. mediation by matrix metalloproteinase ii and matrix metalloproteinase 181. Bromme D and Lecaille F. Cathepsin k inhibitors for osteoporosis and ix. Bioconjug Chem. 2004; 15: 931-41. potential off-target effects. Expert Opin Invest Drugs. 2009; 18: 585-600. 161. Chau Y, Dang NM, Tan FE, et al. Investigation of targeting mechanism of 182. Costa AG, Cusano NE, Silva BC, et al. Cathepsin k: Its skeletal actions new dextran-peptide-methotrexate conjugates using biodistribution and role as a therapeutic target in osteoporosis. Nat Rev Rheumatol. study in matrix-metalloproteinase-overexpressing tumor xenograft 2011; 7: 447-56. model. J Pharm Sci. 2006; 95: 542-51. 183. Sinha AA, Wilson MJ, Gleason DF, et al. Immunohistochemical 162. Alley SC, Okeley NM, and Senter PD. Antibody-drug conjugates: localization of cathepsin-b in neoplastic human prostate. Prostate. 1995; Targeted drug delivery for cancer. Curr Opin Chem Biol. 2010; 14: 26: 171-8. 529-37. 184. Bongers V, Konings CH, Grijpma AM, et al. Serum proteinase activities 163. Senter PD. Potent antibody drug conjugates for cancer therapy. Curr in head and neck squamous cell carcinoma patients. Anticancer Res. Opin Chem Biol. 2009; 13: 235-44. 1995; 15: 2763-6. 164. Doronina SO, Toki BE, Torgov MY, et al. Development of potent 185. Lopez Couto E, Tersariol ILS, Pinhal MaS, et al. Cathepsin b and monoclonal antibody auristatin conjugates for cancer therapy. Nat heparanase activity and expression in head and neck squamous cell Biotechnol. 2003; 21: 778-84. carcinoma. Oral Oncol. 2007;: 96. 165. [Internet] FDA. Fda approves adcetris to treat two types of lympoma. 186. Roshy S, Sloane BF, and Moin K. Pericellular cathepsin b and malignant http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ progression. Cancer Metastasis Rev. 2003; 22: 271-86. ucm268781.htm. 187. Zhang H, Fu T, Mcgettigan S, et al. Il8 and cathepsin b as melanoma 166. Vasey PA, Kaye SB, Morrison R, et al. Phase I clinical and serum biomarkers. Int J Mol Sci. 2011; 12: 1505-18. pharmacokinetic study of pk1 [n-(2-hydroxypropyl)methacrylamide 188. Hara H, Friedlander RM, Gagliardini V, et al. Inhibition of interleukin 1 copolymer doxorubicin]: First member of a new class of beta converting enzyme family proteases reduces ischemic and chemotherapeutic agents-drug-polymer conjugates. Cancer research excitotoxic neuronal damage. Proc Natl Acad Sci U S A. 1997; 94: campaign phase I/II committee. Clin Cancer Res. 1999; 5: 83-94. 2007-12. 167. Seymour LW, Ferry DR, Anderson D, et al. Hepatic drug targeting: 189. Schellmann N, Deckert PM, Bachran D, et al. Targeted enzyme prodrug Phase I evaluation of polymer-bound doxorubicin. J Clin Oncol. 2002; 20: therapies. Mini Rev Med Chem. 2010; 10: 887-904. 1668-76. 190. Martinon F and Tschopp J. Inflammatory caspases: Linking an 168. Duncan R. Development of hpma copolymer-anticancer conjugates: intracellular innate immune system to autoinflammatory diseases. Cell. Clinical experience and lessons learnt. Adv Drug Deliv Rev. 2009; 61: 2004; 117: 561-74. 1131-48. 191. Huttunen KM and Rautio J. Prodrugs - an efficient way to breach delivery and targeting barriers. Curr Top Med Chem. 2011; 11: 2265-87.

http://www.thno.org Theranostics 2012, 2(2) 177

192. Hartmann A, Hunot S, Michel PP, et al. Caspase-3: A vulnerability factor 217. Carter PJ and Gazzard L. Caspase activated prodrugs therapy. Wip and final effector in apoptotic death of dopaminergic neurons in organization. 2001. parkinson's disease. Proc Natl Acad Sci U S A. 2000; 97: 2875-80. 218. Kuefner U, Lohrmann U, Montejano YD, et al. 193. Li MW, Ona VO, Guegan C, et al. Functional role of caspase-1 and Carboxypeptidase-mediated release of methotrexate from methotrexate caspase-3 in an als transgenic mouse model. Science. 2000; 288: 335-9. alpha-. Biochemistry. 1989; 28: 2288-97. 194. Huttunen KM, Raunio H, and Rautio J. Prodrugs--from serendipity to 219. Lo PC, Chen J, Stefflova K, et al. Photodynamic molecular beacon rational design. Pharmacol Rev. 2011; 63: 750-71. triggered by fibroblast activation protein on cancer-associated fibroblasts 195. Ona VO, Li MW, Vonsattel JPG, et al. Inhibition of caspase-1 slows for diagnosis and treatment of epithelial cancers. J Med Chem. 2009; 52: disease progression in a mouse model of huntington's disease. Nature. 358-68. 1999; 399: 263-7. 220. Janssen S, Jakobsen CM, Rosen DM, et al. Screening a combinatorial 196. Lipton P. Ischemic cell death in brain neurons. Physiol Rev. 1999; 79: peptide library to develop a human glandular kallikrein 2-activated 1431-568. prodrug as targeted therapy for prostate cancer. Mol Cancer Ther. 2004; 197. Yoshiyama Y, Arai K, Oki T, et al. Expression of invariant chain and 3: 1439-50. pro-cathepsin l in alzheimer's brain. Neurosci Lett. 2000; 290: 125-8. 221. Janssen S, Rosen DM, Ricklis RM, et al. Pharmacokinetics, 198. Johnson MD, Torri JA, Lippman ME, et al. The role of cathepsin-d in the biodistribution, and antitumor efficacy of a human glandular kallikrein 2 invasiveness of human breast-cancer cells. Cancer Res. 1993; 53: 873-7. (hk2)-activated thapsigargin prodrug. Prostate. 2006; 66: 358-68. 199. Losch A, Schindl M, Kohlberger P, et al. Cathepsin d in ovarian cancer: 222. Chen J, Liu TW, Lo PC, et al. "Zipper" molecular beacons: A generalized Prognostic value and correlation with p53 expression and microvessel strategy to optimize the performance of activatable protease probes. density. Gynecol Oncol. 2004; 92: 545-52. Bioconjug Chem. 2009; 20: 1836-42. 200. Tedone T, Correale M, Barbarossa G, et al. Release of the aspartyl 223. Zheng G, Chen J, Stefflova K, et al. Photodynamic molecular beacon as protease cathepsin d is associated with and facilitates human breast an activatable photosensitizer based on protease-controlled singlet cancer cell invasion. FASEB J. 1997; 11: 785-92. oxygen quenching and activation. Proc Natl Acad Sci U S A. 2007; 104: 201. Reynolds MA, Kastury K, Groskopf J, et al. Molecular markers for 8989-94. prostate cancer. Cancer Lett. 2007; 249: 5-13. 224. De Groot FM, De Bart AC, Verheijen JH, et al. Synthesis and biological 202. Adib TR, Henderson S, Perrett C, et al. Predicting biomarkers for ovarian evaluation of novel prodrugs of anthracyclines for selective activation by cancer using gene-expression microarrays. Br J Cancer. 2004; 90: 686-92. the tumor-associated protease plasmin. J Med Chem. 1999; 42: 5277-83. 203. Yousef GM, Borgono CA, Popalis C, et al. In-silico analysis of kallikrein 225. Carl PL, Chakravarty PK, and Katzenellenbogen JA. A novel connector gene expression in pancreatic and colon cancers. Anticancer Res. 2004; linkage applicable in prodrug design. J Med Chem. 1981; 24: 479-80. 24: 43-52. 226. Chakravarty PK, Carl PL, Weber MJ, et al. Plasmin-activated prodrugs 204. Lu KH, Patterson AP, Wang L, et al. Selection of potential markers for for cancer chemotherapy. 1. Synthesis and biological activity of epithelial ovarian cancer with gene expression arrays and recursive peptidylacivicin and peptidylphenylenediamine mustard. J Med Chem. descent partition analysis. Clin Cancer Res. 2004; 10: 3291-300. 1983; 26: 633-8. 205. Iacobuzio-Donahue CA, Ashfaq R, Maitra A, et al. Highly expressed 227. Carl PL, Chakravarty PK, Katzenellenbogen JA, et al. Protease-activated genes in pancreatic ductal adenocarcinomas. Cancer Res. 2003; 63: "prodrugs" for cancer chemotherapy. Proc Natl Acad Sci U S A. 1980; 77: 8614-22. 2224-8. 206. Chung CH, Parker JS, Karaca G, et al. Molecular classification of head 228. Balajthy Z, Aradi J, Kiss IT, et al. Synthesis and functional evaluation of a and neck squamous cell carcinomas using patterns of gene expression. peptide derivative of 1-beta-d-arabinofuranosylcytosine. J Med Chem. Cancer Cell. 2004; 5: 489-500. 1992; 35: 3344-9. 207. Yousef GM, Scorilas A, Katsaros D, et al. Prognostic value of the human 229. Devy L, De Groot FM, Blacher S, et al. Plasmin-activated doxorubicin kallikrein gene 15 expression in ovarian cancer. J Clin Oncol. 2003; 21: prodrugs containing a spacer reduce tumor growth and angiogenesis 3119-26. without systemic toxicity. FASEB J. 2004; 18: 565-7. 208. Yousef GM, Scorilas A, Jung K, et al. Molecular cloning of the human 230. De Groot FM, Loos WJ, Koekkoek R, et al. Elongated multiple electronic kallikrein 15 gene (klk15). J Biol Chem. 2001; 276: 53-61. cascade and cyclization spacer systems in activatible anticancer 209. Trengove NJ, Stacey MC, Macauley S, et al. Analysis of the acute and prodrugs for enhanced drug release. J Org Chem. 2001; 66: 8815-30. chronic wound environments: The role of proteases and their inhibitors. 231. Wong BK, Defeo-Jones D, Jones RE, et al. Psa-specific and Wound Repair Regen. 1999; 7: 442-52. non-psa-specific conversion of a psa-targeted peptide conjugate of 210. Rosenberg GA. Matrix metalloproteinases in neuroinflammation. Glia. doxorubicin to its active metabolites. Drug Metab Dispos. 2001; 29: 313-8. 2002; 39: 279-91. 232. Kumar SK, Williams SA, Isaacs JT, et al. Modulating paclitaxel 211. Egeblad M and Werb Z. New functions for the matrix metalloproteinases bioavailability for targeting prostate cancer. Bioorg Med Chem. 2007; 15: in cancer progression. Nat Rev Cancer. 2002; 2: 161-74. 4973-84. 212. Heppner KJ, Matrisian LM, Jensen RA, et al. Expression of most matrix 233. Defeo-Jones D, Brady SF, Feng DM, et al. A prostate-specific antigen metalloproteinase family members in breast cancer represents a (psa)-activated vinblastine prodrug selectively kills psa-secreting cells in tumor-induced host response. Am J Pathol. 1996; 149: 273-82. vivo. Mol Cancer Ther. 2002; 1: 451-9. 213. Peake NJ, Khawaja K, Myers A, et al. Levels of matrix metalloproteinase 234. Brady SF, Pawluczyk JM, Lumma PK, et al. Design and synthesis of a (mmp)-1 in paired sera and synovial fluids of juvenile idiopathic arthritis pro-drug of vinblastine targeted at treatment of prostate cancer with patients: Relationship to inflammatory activity, mmp-3 and tissue enhanced efficacy and reduced systemic toxicity. J Med Chem. 2002; 45: inhibitor of metalloproteinases-1 in a longitudinal study. Rheumatology. 4706-15. 2005; 44: 1383-9. 235. Dubois V, Dasnois L, Lebtahi K, et al. Cpi-0004na, a new extracellularly 214. Molloy KJ, Thompson MM, Jones JL, et al. Unstable carotid plaques tumor-activated prodrug of doxorubicin: In vivo toxicity, activity, and exhibit raised matrix metalloproteinase-8 activity. Circulation. 2004; 110: tissue distribution confirm tumor cell selectivity. Cancer Res. 2002; 62: 337-43. 2327-31. 215. Chen J, Stefflova K, Niedre MJ, et al. Protease-triggered photosensitizing 236. Fernandez AM, Van Derpoorten K, Dasnois L, et al. beacon based on singlet oxygen quenching and activation. J Am Chem N-succinyl-(beta-alanyl-l-leucyl-l-alanyl-l-leucyl)doxorubicin: An Soc. 2004; 126: 11450-1. extracellularly tumor-activated prodrug devoid of intravenous acute 216. Stefflova K, Chen J, Marotta D, et al. Photodynamic therapy agent with a toxicity. J Med Chem. 2001; 44: 3750-3. built-in apoptosis sensor for evaluating its own therapeutic outcome in 237. Pan C, Cardarelli PM, Nieder MH, et al. Cd10 is a key enzyme involved situ. J Med Chem. 2006; 49: 3850-6. in the activation of tumor-activated peptide prodrug cpi-0004na and

http://www.thno.org Theranostics 2012, 2(2) 178

novel analogues: Implications for the design of novel peptide prodrugs macromolecular polymer-drug conjugate. Int J Nanomedicine. 2006; 1: for the therapy of cd10+ tumors. Cancer Res. 2003; 63: 5526-31. 375-83. 238. Trouet A, Passioukov A, Van Derpoorten K, et al. Extracellularly 259. O'brien ME, Socinski MA, Popovich AY, et al. Randomized phase III trial tumor-activated prodrugs for the selective chemotherapy of cancer: comparing single-agent paclitaxel poliglumex (ct-2103, ppx) with Application to doxorubicin and preliminary in vitro and in vivo studies. single-agent gemcitabine or vinorelbine for the treatment of ps 2 patients Cancer Res. 2001; 61: 2843-6. with chemotherapy-naive advanced non-small cell lung cancer. J Thorac 239. Dubois V, Nieder M, Collot F, et al. Thimet oligopeptidase (ec 3.4.24.15) Oncol. 2008; 3: 728-34. activates cpi-0004na, an extracellularly tumour-activated prodrug of 260. Paz-Ares L, Ross H, O'brien M, et al. Phase III trial comparing paclitaxel doxorubicin. Eur J Cancer. 2006; 42: 3049-56. poliglumex vs docetaxel in the second-line treatment of non-small-cell 240. Choi Y, Weissleder R, and Tung CH. Selective antitumor effect of novel lung cancer. Br J Cancer. 2008; 98: 1608-13. protease-mediated photodynamic agent. Cancer Res. 2006; 66: 7225-9. 261. Dipaola RS, Rinehart J, Nemunaitis J, et al. Characterization of a novel 241. Choi Y, Weissleder R, and Tung CH. Protease-mediated phototoxicity of prostate-specific antigen-activated peptide-doxorubicin conjugate in a polylysine-chlorin(e6) conjugate. Chem Med Chem. 2006; 1: 698-701. patients with prostate cancer. J Clin Oncol. 2002; 20: 1874-9. 242. Fiehn C, Kratz F, Sass G, et al. Targeted drug delivery by in vivo coupling to endogenous albumin: An albumin-binding prodrug of methotrexate (mtx) is better than mtx in the treatment of murine collagen-induced arthritis. Ann Rheum Dis. 2008; 67: 1188-91. 243. Pan H, Liu J, Dong Y, et al. Release of prostaglandin e(1) from n-(2-hydroxypropyl)methacrylamide copolymer conjugates by bone cells. Macromol Biosci. 2008; 8: 599-605. 244. Pan H, Kopeckova P, Wang D, et al. Water-soluble hpma copolymer--prostaglandin e1 conjugates containing a cathepsin k sensitive spacer. J Drug Target. 2006; 14: 425-35. 245. Pan H, Sima M, Kopeckova P, et al. Biodistribution and pharmacokinetic studies of bone-targeting n-(2-hydroxypropyl)methacrylamide copolymer-alendronate conjugates. Mol Pharm. 2008; 5: 548-58. 246. Kratz F, Drevs J, Bing G, et al. Development and in vitro efficacy of novel mmp2 and mmp9 specific doxorubicin albumin conjugates. Bioorg Med Chem Lett. 2001; 11: 2001-6. 247. Warnecke A, Fichtner I, Sass G, et al. Synthesis, cleavage profile, and antitumor efficacy of an albumin-binding prodrug of methotrexate that is cleaved by plasmin and cathepsin b. Arch Pharm (Weinheim). 2007; 340: 389-95. 248. Gabriel D, Busso N, So A, et al. Thrombin-sensitive photodynamic agents: A novel strategy for selective synovectomy in rheumatoid arthritis. J Control Release. 2009; 138: 225-34. 249. Hamblin MR, Miller JL, Rizvi I, et al. Pegylation of a chlorin(e6) polymer conjugate increases tumor targeting of photosensitizer. Cancer Res. 2001; 61: 7155-62. 250. Hamblin MR, Miller JL, Rizvi I, et al. Pegylation of charged polymer-photosensitiser conjugates: Effects on photodynamic efficacy. Br J Cancer. 2003; 89: 937-43. 251. Gabriel D, Campo MA, Gurny R, et al. Tailoring protease-sensitive photodynamic agents to specific disease-associated enzymes. Bioconjug Chem. 2007; 18: 1070-7. 252. Campo MA, Gabriel D, Kucera P, et al. Polymeric photosensitizer prodrugs for photodynamic therapy. Photochem Photobiol. 2007; 83: 958-65. 253. Chung DE and Kratz F. Development of a novel albumin-binding prodrug that is cleaved by urokinase-type-plasminogen activator (upa). Bioorg Med Chem Lett. 2006; 16: 5157-63. 254. Jeffrey SC, Nguyen MT, Andreyka JB, et al. Dipeptide-based highly potent doxorubicin antibody conjugates. Bioorg Med Chem Lett. 2006; 16: 358-62. 255. Francisco JA, Cerveny CG, Meyer DL, et al. Cac10-vcmmae, an anti-cd30-monomethyl auristatin e conjugate with potent and selective antitumor activity. Blood. 2003; 102: 1458-65. 256. Hopewel JW, Duncan R, Wilding D, et al. Preclinical evaluation of the cardiotoxicity of pk2: A novel hpma copolymer-doxorubicin-galactosamine conjugate antitumour agent. Hum Exp Toxicol. 2001; 20: 461-70. 257. Galis ZS and Khatri JJ. Matrix metalloproteinases in vascular remodeling and atherogenesis - the good, the bad, and the ugly. Circ Res. 2002; 90: 251-62. 258. Chipman SD, Oldham FB, Pezzoni G, et al. Biological and clinical characterization of paclitaxel poliglumex (ppx, ct-2103), a

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